EXPRESSION, PURIFICATION, REFOLDING, AND ATP BINDING OF THE FIRST NUCLEOTIDE BINDING

DOMAIN OF THE CYSTIC FIBROSIS /

TRANSMEMBRANE CONDUCTANCE REGULATOR --

Ronald Thomas Grondin

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Molecuiar and Medical Genetics University of Toronto

O Copyright by Ronald Thomas Grondin, 1997

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Abstract

Expression, Purification, Refolding, and ATP Binding of the First

Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane

Conductance Regulator. Ronald Thomas Grondin, Master of Science, 1997.

Graduate Department of Molecular and Medical Genetics, University of

Toronto.

Cystic Fibrosis (CF) is an autosomal recessive disorder affecting

approximately 1 in 2500 Caucasians. CF is caused by mutations in the Cystic

Fibrosis Transmembrane Conductance Regulator (CFTR), a chloride channel

involved in ion and fluid transfer across the apical membrane of epithelial

cells.

To study the functional effects of mutations in the first nucleotide

binding dornain (NBD1) of CFTR, a collection of NBDl peptides were

constructed and expressed in E. coli. These peptides were purified, refolded,

and assessed for their ability to bind ATP and ATP analogues. Despite the

inherent insolubility encountered with NBD1, soluble peptides were

eventuafly obtained by refolding in the presence of high concentrations of L-

arginine (400 mM). Three peptides, corresponding to amino acids 404-589, 384-

649, and 415-675, were capable of refolding into monomeric species and

displayed CO-operative stability. However, the non-physiological conditions

required to maintain the monomeric state resulted in a loss of measurable

ability to bind ATP.

Acknowledgements

In the final hours (and minutes) of completing this thesis, it is my

priviledge to thank those who have contributed so much of their time and

energy, not only to the substance of the this project, but also to my

development as a scientist. 1 am greatly indebted to my supervisor, Dr.

Johanna M. Rommens for her advice, enthusiasrn, and encouragement at

every stage of my graduate education. 1 shall recall her endless supply of

energy (and interesting stories) well into the future.

1 also wish to thank the past and present rnembers of the Rommens lab

and of the 11th floor with whom I have had the opportunity to work, and to

play. 1 wish them al1 the best of success in their careers, and happiness i n

their personal lives.

1 wish to acknowledge our collaborators, Dr. Julie Forman-Kay, and Dr.

Henry Mok, with whom much of the later stages of this work was done. 1

also wish to acknowledge Dr. Brenda Andrews and Dr. Peter Ray for their

guidance throughout this project.

Finally, 1 wish to thank my family, and especially my parents, for their

support and encouragement throughout every stage of my life.

iii

Table of Contents

.. Abstract ......................................................................................... I I

... ........................................................................... Acknowledgements 1 1 1

............................................................................ Table of Contents i v

. . .............................................................................. List of Figures v I i

... .............................................................................. List of Tables v 1 1 1

....................................................................... List of Abbreviations i x

.................................................................................... Introduction 1

Cystic Fibrosis ............................................................................................................. 1 ...................................................... 1.1. Respiratory Tract Abnorinalities irz Cysric FiOrosis 1

........................................................................ 1.2. Po~icrmtic Disecise itz Cystic Fibrosis -2 .................................... 1.3. Other Clitzical Manijestarions of Cystic Fibrosis : ...................... 3

1.3. Treatnrent .............................................................................................................. 4 Structure of CFTR ........................................................................................................ 4 2.1. ABC Sicpctj4amily ................................................................................................... 4 2.2. CFTR is rnottonzeric .............................................................................................. 7

............................................................................................................... Thc CF Gene 8 .................................................................................... 3.1. Identification of tlze CF Gerle 8

....................................................................... 3.2. Evolictiotzary Cortservation .......... .. 9 ................................................................................ 3.3. Identification of CF Mrctations I O

3.4. The Mirrine Ortlzolog of CFTR arid Mortse ~Models for CF ........................................... IO 3.5. Gerte Tlzerapy ...................................................................................................... I l

...................................................................................................... Ftinction of CFTR 12 ............................................................................................ 4.1. Chloride Conductance 12

............................................................. 4.1.1. Regulation of CFTR Chloride Conductance 13 ......................................................................... 4.1.1.1. R Domain Phospliorylation 13

4.1.1.2. ATP BindingIHydrolysis ............................................................................ 15 ................................................................. 4.1.2. Features of the CFTR Chloride Channel 16

...................................................................................... 4.1.2.1. Thrce state mode1 1 6 .......................... .................................. 4.1.2.2. Double occupancy of the ion pore ..,. 17

................................................................................. 4.2. Regiclation of Other Chantzels 18 4.2.1. Outwardly-Rectifying Chloride Channels (ORCC) .................................................... 18 4.2.2. ATP Conductance ........................................................................................... 19

......................................................................................... 4.2.3. Sodium Channels -20 ......... 4.3. Regielation of otlzer ceil functions: pH. sialylntiorr. mld pinnna mnibrane recycling 22

....................................................................................................... . 3 CFTR Processing 23 .......................................................................... . . 5 1 CFTR Exl~rcssiorz uttd Localisatiori 23

...................................................................................................... 5.2. C~~cosyiat io~i 24 .................................................................................. 5.3. Assaciatiotr with Cltaperones 25 ................................................................................. 5.4. P o o r Eflciericy of Processirzg 26

............................................................................................... 5.5. CFTR Degradution 27 6 . CFTR Mutations ........................................................................................................ 27

................................................................................... . . 6 / Classes of CFTR Mi~tarions 27 .............................................................................. 6.1 . 1 . Dc fcctive Protein Production 27

........................................................................................ 6.1.3. Defective Regulation 2 8 ...................................................................................... 61.4. Defective Conductance 29

........................................................................................ 6.1 .S. Alternative Splicing 29 6.2. AF508 is a Processing Mittution ............................................................................. 29

.......................................................................................... 6.3. Otlîer NBD Mutations 3 1 ............................................................................................................ 7 . NBD Studies . .33

.............................................................. 8 . NBDl Expression, Purification and ATP Binding 34

...................................................................... Materials and Methods 3 6

.............................................................................................. . 1 NBD Expression Vectors 36 ..................................... 1.1. Clonirtg of NBD1348,, , into the pPINPOINT expression vector 36

1.2. Cloning of NBD I,,,. NBDI,.,5. and NBDl/R,,.,8 into the PET expression vector ..... 3 6 1.3. Cloning of NBD14,.5,,. NBD I ,,.,, and NBDI,,.,, irlto the pET expression vector ......... 38

...................................................... 1.3. Cumtruction of Vectors Erzcoding NBD1 prptide.~ 39 .................................................... . 2 Expression. purification. and refolding o f N B D 1 peptides 40

2.1. EA'pressiorz. picrifrcation. and refolding of NBDI,,,, expresseci fioin y PINPOINT Vectors 40 ........... 2.2. Expression. pizrijlcation. and refolding of NBDI,.~.,,, Expressed front PET Vectors 41

2.3. Expression. purification. artd refolding of NBDI, ,,,, NBDI ,,.,, alrd NBD 1415.6m .......... 43 2.4. Crmtiidirie denaturation of refolcied peptides ................................................................ 44

....................................................................... 2.5. Li@ Scatsering of Refulded Proteins 44 2.6. Gel filtration of refolded NBDI peptides .................................................................... 45

3 . ATP binding assays ..................................................................................................... 45 ............................................................................................. 3.1. 6-arido-ATP birzding 45

.......................................................................... 3.2. l~itr-itrsic Ttyptoylzutz Fluorescence 4 6 ........................................................................................................... 3.3. TNP-A TP 46

Resrilts and Discussion ..................................................................... 4 7

........................................................................................ . 1 PINBD 1 M..,. Peptide 48 ....................................................... 1.1. fipression and Purrjication of PINBDI Peptides 4 8

........................................................................... 1.2. ATP Bintling of PINBDI Peptides 51 ..................................................................................................... 2 . pETNBD 1 Peptides 54

.......................................................... 2.1. Expression and P~irification of pETNBD13484,, 54 ............................................................... 2.2. pETNBD1 M.7.,,, Aggregates ilporz Refoldirig 56

2.3. pETNBDl.f4.,,y does not birid ATP by citlzer 8-azido-ATP o r TNP-ATP binding assays ..... 56 3 . Oilicr Ai tcmp~s to Find a Soluble NBD 1 Pepiide ............................................................. -57

............................................................................. 3.1. Expr-essiorz of pETNBD1 57

............................................................................. 3.2. Espss io r t of pETNBDI/R ,,,., , 58 ............................................................. 4 . N B D 14(M.5b. NBD I 3X4.(C11). and NBD L , , , , , Peptides 59 ........................................................... 4.1. Expressiorr ar~d Prwification of NBDl pepridcs 62

4.1. NUL) I Peptides Kenzain Soluble and Monor~ieric in the Preserzce of 0.4 M L-Argirrine ...... 63 4.3. NBDI Peptides Refold irtto Co-operatively Foldetl Doniaim ......................................... 65 4.4. ATP Bitidiltg of NBD14,.J,, NBD 1 ,.,, atid NBDI.,,., f i Peptides ................................ 66

................................................................................................ 4.4.1 . 8-azido-ATP -66 4.4.1.1. NBD 1 peptides are labelled by [y.3'P].8.azido.~~~ ............................................ 67 4.4.1.2. The labelling o f NBDl peptides by [a-32~]-8-az ido-~TP is stronger tlian that by

.................................................................................. [~."P].~.~z~~o.ATP 7 0 4.4.1.3. ATP does not compeie for 8-azido-ATP bindiiig ................................................ 71 4.4.1.4. [ W 3 2 ~ ~ - 8 - a z i d o - ~ ~ ~ binds to aggrcgated proirins ............................................. 7 3

....................................................................... 4.4.2. lntrinsic Tryptophan Fluorescence 7 5 4.4.3. TNP.ATP ....................................................................................................... 7 6

............................................................................... 4.5. Aggr-egtitiori of N13DI Pcpicles 79 4.5.1. 1-ligli Concciilralions o f L-Arginine Prcvciii Agg~.cg.iiion of N I 3 D I Pcplitlcs .................... 7 9 4.5.2. NBD 1 Pcptides arc Icss solublc at lowcr p H ............................................................ 8 2

Conclustons and Future Directions ....................................................... 15 3

1 . L-Arginine Prevents Protein Aggregation ........................................................................ 83 ........................................................................................... 2 . Correct Domain boundaries 84

3 . NBDI extends into the putative R domain ....................................................................... 85 4 . ATP Binding .............................................................................................................. 86

........................................................................................................... 5 . Protein I'olding 87

.................................................................................. Appendices 108

1 . Appendix A . Raw Data for Light Scattering of NBD 1 Peptides at different pH ..................... 108 2 . Appendix B . Raw Data of Light Scattering of NBDl Peptides Refolded in Varying

................................................................... Concentrations of L.Arginine Il1 ...................................... 3 . Appendix C . Effect of pH on Light Scattering oFNBDl Peptides 119

.............. 4 . Appendix D . Raw Data of the Guanidium Chloride Dcnaturation of NBD1 Peptides 120 ............................................ 5 . Appendix E . Raw Data of TNP-ATP Binding to NBDI ,,,,,,, 121

.................................... 6 . Appcndix F . Raw Data of TNP-ATP Binding to Adenylate Kinase 122

. Figure 1 . Model of CFTR ................................................................................................... G

............................................... Figure 2 . Sequence of Synthetic Oligonucleotide Inscrt 37

Figure 3 . NBDl Constructs ............................................................................................. 48

Figure 4 . Expression of pPINBD1-wt .............................................................................. 49

................................................................. Figure 5 . Partially Purified PINBDl proteins 51

.................................. Figure 6 . [a-32P]-8-azido-ATP Binding to PlNBDl Crude Extract 52

Figure 7 . [a-3'P]-8-azido-ATP Binding to Partially Purified PINBDl Fusion Peptides.53

Figure 8 . Expression of pETNBDI3.... ... .......................................................................... 54

Figure 9 .Purification of pETNBDl,,,.,,,. .......................................................................... 55

Figure 10 . Expression of pETNBD11 R34n.nsii ............................................................... 5 9

Figure 11 . Expression and Purification of NBDI,,,,.,, , and NBDl,,, .,,, ............................. 62

............................................................................ Figure 12 . Gel Filtration of NBDI,,,., , , 64

Figure 13 . Guanidinium Chloridc Dcnaturation of NBDl Peptides ................................ 65

Figure 14 . [y-32P]-8-azido-ATP binding to NBDl.... ...Y...................................................... 68

Figure 15 . [y-3tP]-8-azido-ATP binding to NBD13 84.64. C542A and C524S ...................... 69

Figure 16 . Characterisation of the labelling of N13D13n4.649 by [a.32P].8.azido.~TP ...... 71

............. Figure 17 . Conipetition of [ a - 3 2 P ] - 8 - a z i d o - ~ ~ ~ labelling of NBDl with ATP 72

Figure 18 . Non-specific Labelling of Proteins by [a-32P]-8-azido-ATP ......................... 72

Figure 19 . [~x-~~P]-S-azido-ATP Labelling of Protein Aggregates ................................... 74

Figure 20 . Autoradiograph of [a-32P]-8-azido-ATP binding to NBDl, 1s.6.s...................... 75

Figure 21 . TNP-ATP Binding to NBDl,,,,.,,, ..................................................................... 77

Figure 22 . Effcct of L-Arginine on TNP-ATP Binding to Adcnylate Kinase ................ 79

Figure 23 . Liglit Scattering of NBDl,,,, .,,, ........................................................................ 80

Figure 24 . Light Scattering of NBDI,,,.,,, ........................................................................ 81

Figure 25 . Liglit Scattering of NBDI,,,., , , ........................................................................ 81

Figure 26 . Effect of pH on the Liglit Scattering of NBD13e4.r49. ..................................... 82

Table 1 . l'CR Prirners for NBDI Peptides .................................................................... 39

........................... Table 2 . IJCR Priniers for C491A/S and C524AlS NBDl Mutations 40

....................................................... Table 3 . Kefolding Conditions for pETNBD1348,yy -42

viii

List of Abbreviations

aa

ATP

CAMP

cDNA

CFTR

CFTR-wt

cRNA

DIDS

DPC

DTT

ECL

EDTA

ER

G551D

GdmCl

HOAc

HRP

IPTG

MDS

NBD

ORCC

PKA

PKC

SDS-PAGE

AF50S

amino acid

adenosine-5'-triphosphate

cyclic adenosine-5',3'-monophosphate

complementary deoxyribonucleic acid

cystic fibrosis transmembrane conductance regulator

wild type CFTR protein

complimentary ribonucleic acid

4,4'-diisothiocyanotostilene-2,2'-disulphonc acid (ORCC

selective inhibitor)

diphenylamine-2-carboxylic acid (chloride channel inhibitor)

dithiothreitol

enhanced cherniluminescence

ethylenediaminetetracetic acid

endoplasmic reticulum

substitution of glycine with aspartic acid at aa position 551

guanidinium chloride

acetic acid

horseradish peroxidase

isopropyl-P-D-thiogalactoside

membrane spanning domain

nucleotide binding domain

outwardly rectified chloride channel

protein kinase A

protein kinase C

sodium dodecyl sulphate - polyacrylamide gel electrophoresis

deletion of phenylalanine at aa position 508

Introduction

1. Cystic Fibrosis

Cystic fibrosis (CF) is the most common fatal autosomal recessive

disorder affecting various Caucasian populations, with an occurrence of 1 i n

2000 to 3000. I t is caused by mutations in the CF gene, of which more than 700

have been identified (Tsui, 1994-1997). The CF gene encodes the Cystic Fibrosis

Transmernbrane Conductance Regulator (CFTR), a chloride channel involved

in ion and fluid transport across the apical membrane of epithelial cells. The

most common mutation, AF508, accounts for approximately 68% of disease

alleles world-wide and results in a failure of mature protein to reach the apical

surface of epithelial cells. Consequently, CF epithelial tissue is defective i n

electrolyte transport leading to a number of clinical features, some of which

are described below.

1.1. Respiratory Tract Abnormalities in Cystic Fibrosis

Chronic pulmonary obstruction and infection is the principal

contributor to patient mortality in CF and is characterized by a viscous

obstruction of mucous in the bronchioles accompanied by bronchiolar wall

inflammation (Welsh et al., 1992; Zuelzer and Newton, 1949). The bacterial

pathogens Stnphylocucciis a u r e m and Psezidonronns nerzrginosn are

commonly isolated frorn respiratory tract secretions in CF patients. A mucoid

often associated with rapid pulmonary deterioration (Simmonds et al., 1990).

Undigested DNA from dead epithelial cells appears to contribute to the

viscosity of the mucous secretions; when nebulized DNase 1 is introduced

into the airways, it is accompanied by reduction of the viscosity of the mucous

and clinical improvement in many patients (Aitken et al., 1992; Ranasinha et

al., 1993).

The inability to achieve efficient rnucociliary clearance probably

contributes to an impairment in the mechanical defence against lung

infection. However, it is also possible that surface properties in CF epithelia

may be altered in such a way that bacterial adhesion to airway epithelia may

be improved, thus resulting in more persistent infections. Such changes

could include altered presentations of ce11 surface proteins and

glycoconjugates. Indeed, a reduction of sialylated membrane glycoconjugates

is seen in cells expressing CFTR-AF508, the most common mutation in the CF

gene (Dosanjh et al., 1994). As a result, once bacterial colonisation occurs, the

lung infection is nearly impossible to eradicate, and leads to a progressive

deterioration in lung function.

1.2. Pancreatic Disease in Cystic Fibrosis

Exocrine pancreatic insufficiency (PI) is clinically apparent in 85% of CF

patients. It is usually present early in life, but may be progressive. PI is often

mai-tifested by steatorlwa and malnutrition. Clinically, PI is diagnosed by

L L L V U L L U A L V L A G P V A A L L U A A U L U A L U A b U U L L U L G V G L 3 V I J G L U I A L C L J Y O A L L V 6 L L L f

pancreatic colipase, and pancreatic trypsin. CF patients with pancreatic

insufficiency usually require dietary enzyme supplements by their seventh

year (Corey et al., 1989). The remaining 15% of CF patients with pancreatic

sufficiency (PS) do not require enzyme supplements, and generally have a

better prognosis, although individuals rnay not show improved lung

function over PI patients.

It has been demonstrated that pancreatic function is genetically

determined by two subpopulations of CF mutations (Kerem et al., 1989;

Kristidis et al., 1992). Pancreatic sufficiency arises when at least one of the two

mutant alleles is a mild mutation, thereby providing a threshold level of

CFTR function at least in some tissues. It is of interest to note that with the

exception of one splicing mutation, al1 PS mutations known to date are

missense mutations occurring in the transmembrane domains or in the first

nucleotide binding domain of CFTR.

1.3. Other Clinical Manifestations of Cystic Fibrosis

Other tissues affected by CF include epithelial tissue in the

gastrointestinal and genitourinary tracts and in sweat glands. Consequently,

10-20% of new-borns with CF are born with meconium ileus, a viscid

obstruction in the ileum. This complication is life tl~reatening and must be

treated either with an enema that is refluxed into the terminal ileum or, if

this fails, with surgery.

-A & A & A L - & k, 6 A L U L L A LA L C O / V IV V A A A . H A b Y M L L A A & * L A L I A b , V L L V A .-UA J '-

maldevelopment of the vas deferens, epididymus, and seminal vesicles

(Welsh et al., 1992).

The most consistent abnormality in CF patients is elevated levels of

sweat electrolytes, a result of the failure of sweat glands io reabsorb chloride

salts. It is this manifestation which is used to diagnose CF. It is interesting to

note that this same observation, that of salty sweat, had in fact been associated

with early demise of children centuries before cystic fibrosis was first reported

(Welsh et al., 1992).

1.4. Treatment

The treatment of CF has not as of yet been directed at correcting the

genetic defect, but rather at relieving some of the most serious clinical

syrnptoms mentioned above. Such treatments include postural drainage

with chest percussion, aggressive administration of antibiotics, and a

nutritional regimen including pancreatic enzymes and fat-soluble vit amins.

These treatments have extended the mean survival age of CF patients from

early in childhood to approximately 29 years (Welsh, et al., 1992).

2. Structure of CFTR

2.1. ABC Superfamily

CFTR belongs to a large group of membrane transport proteins, known

as the ATP-Binding Cassette (ABC) Superfamily. This group of pro teins

human proteins, MDRl and MRP, involved in multiple drug resistance to

cytotoxic agents during chemotherapy of cancer cells, 2) the STE6 protein,

involved in transport of yeast mating factor, and 3) the bacterial proteins,

MalK and HisP, involved in nutrient transport. The complete sequence of

the budding yeast genome has revealed 29 genes which may encode proteins

that belong to this superfamily (Decottignies and Goffeau, 1997).

Members of the ABC superfamily are similar both structurally and i n

many cases, functionally. Most of them are transporters possessing one or

two membrane spanning domains (MSD) and one or two nucleotide binding

domains (NBD), containing the highly conserved Walker A, B, and C

signatures (Michaelis and Berkower, 1995). The domains are usually

alternating, when they are present in the same polypeptide, but may also be

bipartite and encoded by different polypeptides.

CFTR is distinct from the other members of the ABC superfamily i n

two ways. First, it is a CAMP-gated chloride channel rather than an active

pump that transports via a stoichiometric mechanism. Second, CFTR

possesses a unique domain, the R domain, whose phosphorylation is

involved in regulation of chloride channel activity.

The structural mode1 for CFTR has been based primarily on alignment

with other members of the ABC superfamily (Hyde et al., 1990; Riordan et al.,

1989). The predicted topology of CFTR is shown in Figure 1. As indicated, the

two riucleotide binding domains (NBDI and NBD2) a s well a s the regulatory

\--, --'"'"'" -- - -- ---- -- -, --r----- ------, - ----- ---" " -A'- ""'-"'- r -------- an N-terminal membrane spanning domain (MSD) which together define the

channel pore. This topology corresponds to that of Cluster II in the

phylogenetic classification of yeast ABC proteins (Decottignies and Goffeau,

1997) with the inclusion of the R domain bet-ween the two homologous

halves. Based on alignment, the tertiary structure of the NBDs has been

predicted to be similar to the structure of adenylate kinase (Hyde et al., 1990)

and FI-ATPase (Annereau et al., 1997).

Figure 1. Mode1 of CFïR.

The proposed membrane topology of CFTR has been experimentally

verified by glycosylation site insertion (Chang et al., 1994). By this method,

only extracellularly located N-glycosylation sequences (NXS/T) can be

glycosylated by the transfer from dolichol pyrophosphate (DPP), located in the

asparagines 894 and 900, in the first extracellular loop of MSD2.

Although direct experimental evidence for the structure of CFTR is

somewhat limited, inferences can be made from experimental data from one

of its close relatives, P-glycoprotein (MDR1). Associations have been

demonstrated between domains of human P-glycoprotein using co-

immunoprecipitation assays (Loo and Clarke, 1995). Interactions between the

two halves of P-glycoprotein are mediated by associations between the two

MSDs as well as the two NBDs. In addition, the NBD associates with the MSD

in each l-ialf of the molecule.

The three dimensional architecture of P-glycoprotein has recently been

observed at 2.5 nm resolution by electron microscopy and image analysis

(Rosenberg et al., 1997). The protein, when viewed from above, has a six-fold

symmetry and a diameter of about 10 nm with a 5 nm pore. Its projection

through the membrane has a maximum depth of 8 nm with two 3 nm lobes

on the cytoplasmic surface, likely corresponding to the two NBDs. Labelling

with lectin-gold particles suggest that p-glycoprotein is monomeric.

2.2. CFTR is monomeric

The observation that P-glycoprotein is monomeric has been extended

to CFTR by two different studies. First, mutant variants of CFTR and

monoclonal antibodies were used to study CFTR in several different ce11

types. In this study, CFTR was detected exclusively as a monomer (Marshall

the R domain) is capable of forming a regulated chloride channel (Sheppard

et al., 1994). Single channels were detected whose conductive properties were

comparable to those of wild type CFTR, but did not require R domain

phosphorylation for activation. However, R domain phosphorylation

enhanced activity of CFTR. In sucrose gradient centrifugation of truncated

protein preparations, CFTR elutes at a molecular weight consistent with full

length protein, suggesting that the structure of CFTR may be similar to that of

3. The CF Gene

3.1. Identification of the CF Gene

The CF gene was mapped to chromosome 7q31.2 and was first isolated

in 1989 by positional cloning (Riordan et al., 1989; Rommens et al., 1989;

Kerern et al., 1989) . It consists of 27 exons encoding 1480 amino acids. It was a

significant discovery at the time, not only for the course of CF research, but

also because it marked the first time that a gene was cloned without any

previous knowledge of the biochemical defect or with the benefit of

chromosomal alterations.

The genomic sequence of the CF gene was determined (Zielenski et al.,

1991) and colinearity with the CFTR cDNA was established. Unlike F

glycoprotein, CFTR does not appear to have arisen from a gene duplication

event: an observation based upon differences in the two halves of the gene.

perhaps inserted into an MDR-like precursor of CFTR.

Coinparison to P-glycoprotein has been further explored with the

finding that P-glycoprotein may be a volume regulated chloride channel (Bear

and Ling, 1993). Furthermore, expression of p-glycoprotein in various tissues

seems to complement the expression of CFTR (Trezise et al., 1992) suggesting

that CFTR and P-glycoprotein may be functionally related. The potential

functional similarity between CFTR and p-glycoprotein has led to speculatio

that CFTR may be involved in other functions in addition to its chloride

chaiïnel ac tivity.

3.2. Evolutionary Conservation

CFTR has been identified in species as divergent as the spiny dogfish,

Squdils ncnntlzias, with 72% amino acid identity to the human ortholog

(Marshall et al., 1991). In the dogfish, it is also localized apically, in this case

in the rectal gland. There exists even greater conservation in CFTR across

mammalian species, ranging as high as 93% in exon 10 and 82400% in exon

11 (Gasparini et al., 1991). In contrast, the R domain is a region in which there

is relatively low conservation (63%)) with the exception of a series of

conseiîsus PKA and PKC sites (Diamond et al., 1991). This has led to

speculation that the R domain does not possess any conserved structure, but

rather regulates CFTR chloride channel activity by virtue of its overall charge.

More than 700 CF mutations have been identified since the cloning of

the gene (see section 6.1). However, a large proportion of mutations have

been identified in regions corresponding to the two NBDs (Cutting et al., 1990;

Kerem et al., 1990). Many of these mutations occur in regions of very high

conservation, within or near the consensus Walker motifs, supporting their

crucial role in the function of CFTR.

3.4. The Murine Ortholog of CFTR and Mouse Models for CF

Cloning of the mouse ortholog of CFTR (Tata et al., 1991; Yorifuji et al.,

1991) has made possible the study of an animal mode1 for CF. The murine

ortholog of CFTR has 78% overall identity to the human protein, with higher

identity in the MSDs and NBDs (81-87%) and relatively less conservation in

the R domain (69%) (Yorifuji et al., 1991). The most commonly mutated

amino acid residue, F508, is also conserved in the mouse. CF knockout mice,

however, display a different phenotype than that seen in humans. Most

strains of CF mice do not appear to have significant pulmonary disease, but

have a high incidence of intestinal blockage, similar to meconium ileus,

which results in death shortly after birth. There does exist, however, a

subgroup of CFTR(-/-) mice that show prolonged survival, possibly due to the

up regula tion of Ca+* activated chloride conductance in intestinal epithelia

controlled by a second genetic factor (Rozmahel et al., 1996; Wilschanski et al.,

1996).

cause of the relatively high prevalence of CF in Caucasian populations. It has

been postulated that there rnay exist a significant heterozygote advantage i n

resistance to cholera toxin (Rodman and Zamudio, 1991). Cholera toxin

mediates its action by an irreversible elevation in the intracellular

concentration of CAMP, which causes subsequent dehydration and death. It is

believed that this action may be via the chloride channel activity of CFTR.

Accordingly, CF heterozygotes may have an increased resistance to cholera

toxin, by virtue of reduced levels of CFTR. Consistent with this hypothesis, is

the direct correlation of fluid secretion in response to cholera toxin seen in

mice carrying two, one or no copies of the CF gene (Gabriel et al., 1994).

Another interesting observation has been seen in CF mice in which the

hurnan missense mutation, G551D, has been introduced. Unlike CFTR-

AF508, CFTR-G551D appears to be properly processed, but is believed to be

defective in regulation because of an inability to activate the chloride channel

via ATP binding/hydrolysis (discussed in section 6.1). G551D is also

associated with a reduced risk of meconium ileus; this phenotype appears to

be conserved in the mouse mode1 (Delaney et al., 1996).

3.5. Gene Therapy

For most patients, the most serious pathologic feature of CF is chronic

lung infection, resulting in reduced lung function. Because of accessibility,

gene therapy trials have been pursued via aerosol spray into the airway.

successful. The main shortcomings have been two-fold. First, complications

arise due to inflarnmatory response to the adenoviral vectors used in gene

therapy (O'Neal and Beaudet, 1994). Second, even in treatments using

synthetic liposomes where inflammation is not a problem, the gene transfer

is not efficient and only temporary correction of the CF deficit is seen (Caplen

et al., 1995).

However, gene therapy for CF is still in its early stages. As such, there

remains much to be learned about how to effectively and safely express wild

type genes in the cells of affected tissues with lasting effects.

4. Function of CFTR

4.1. Chloride Conductance

CFTR is a low conductance (6-10 pS) chloride channel with a linear

current/voltage (I/V) relationship (Welsh, et al., 1992). When CFTR is

transfected into mouse fibroblast, HeLa, and CHO ce11 lines, a CAMP-inducible

chloride conductance, can be measured by bot11 whole ce11 conductance and

single cl-iai~i-iel patch clamp studies (Anderson et al., 1991b; Rommens et al.,

1991). In contrast, the major CFTR mutant, AF508, is unable to confer

chloride permeability when stably expressed in mammalian ce11 lines.

CFTR has also been expressed in Sf9 cells, using baculoviral vectors

(Kartner et al., 1991). In these cells, functional CFTR can be highly expressed,

and is localized at the ce11 surface and at intracellular membranes. The high

I

reconstitution of CFTR in proteoliposomes, where it has been studied in the

absence of interacting proteins (Bear et al., 1992).

4.1.1. Regulation of CFTR Chloride Conductance

4.1.1.1. R Donzain Phosphoylation

CFTR chloride conductance is regulated by phosphorylation of the R

domain by CAMP-dependant protein kinase A (PKA) (Cheng et al., 1991). R

domain phosphorylation is required for channel activation. The R domain

contains te11 PKA and PKC consensus sites, and may also contain some cryptic

sites. Cheng et al. identified four of these consensus sites (S660, S737, S795,

and S813) which, when mutated to alanine, resulted in no detectable activated

response. However, using a more sensitive measurement, PKA-dependant

activation of a variant of CFTR in which al1 ten of the PKA and PKC

consensus sites are rnutated to alanine (IOSA), is detectable (Chang et al.,

1993). This suggests that other cryptic PKA sites reside within the R domain.

One suc11 site was identified by phosphorylation and peptide mapping

(S753A); mutation of S753 resulted in a further 40% reduction of channel

activation over the lOSA mutant (Seibert et al., 1995).

Not only can chloride channel activation be controlled by the addition

of CAMP-agonists, but phospha tase inhibi tors are also capable of channel

activation in excised membrane patches (Becq et al., 1994). This supports a

mode1 for CFTR in which R domain phosphorylatioi~ is dyi~amic, but

can be maintained.

The R dornain appears to act as a 'plug' for the ion pore, and is

effectively cleared away from the pore upon conformational change by

introduction of negative charge via phosphorylation or mutagenesis. A

CFTR derivate in which the PKA serines are mutated to glutamic acid causes

constitutively open channels (Rich et al., 1993a). The same result is also

obtained when a large portion of the R domain (aa. 70û-835) is deleted (Rich et

al., 1991). However, this deletion mutant also showed residual low level

response to CAMP.

Dulhanty et al. have proposed a two-domain mode1 for the R domain

based on extensive sequence alignment across ten animal species (Dulhanty

and Riordan, 1994a). In this study, they noted that there is a relatively higher

conservation in the first third of the R domain (aa. 587-672). Furthermore,

the majority of the conserved PKA sites occur in the latter two tliirds of the

domain. Based on these observations, it appears that the first part of the R

domain (RD1) may have structural importance, whereas the second part

(RD2) acts through some conformational change associated with

phosphorylation.

Circular dichroism (CD) studies on the secondary structure of the R

domain have revealed that there is a reduction in a-helical content upon

PKA phosphorylation (Dulhanty and Riordan, 1994b). Furtherrnore, there is

a significant change in the CD spectra of the R domain when several PKA

et al., 1995), consistent with the idea that a conformational change in the R

dornain is needed to allow chloride channel activation.

The N-terminal portion of CFTR (up to and including the R domain) is ,. . -

capable of forming a chloride channel with comparable conductance

properties to wild type CFTR, but with altered R domain regulation

(Sheppard et al., 1994). Presumably, this is the result of the formation of a

dimer of half molecules. In this N-terminal derivative of CFTR, R domain

phosphorylation is not necessary for channel activation possibly due to steric

interactions between the R domains of each half molecule.

4.1.1.2. ATP BindingIHydro lysis

Following R domain phosphorylation, a source of hydrolysable,

cytosolic ATP is required to open the CFTR chloride channel (Anderson et al.,

1991a). The ATP requirement reflects the direct binding of ATP to CFTR,

rather thsn an indirect action mediated by anotl~er protein, since CFTR cari be

labelled with Sazido-ATP, an ATP analogue capable of covalent attachment

following UV irradiation (Travis et al., 1993). Millimolar levels of ATP or

GTP are capable of inhibiting this labelling, but AMP is not.

ATP binding/hydrolysis at both NBDs of CFTR is involved in channel

gating, and the two NBDs have different functions. Homologous mutations

in the two NBDs have distinct effects; mutations in NBDl result in reduced

activation, but mutations in NBD2 result in higl-ier open channel probability

(Anderson and Welsh, 1992; Smit et al., 1993 ). Carson et al. analysed the

decreased frequency of bursts of channel activity. However, NBD2 mutations

also resulted in prolonged bursts of activity, thereby increasing the overall

open state probability (Po) (Carson et al., 1995). Since none of the mutants

studied had a reduced ability to bind 8-azido-ATP, the effects were likely the

result of a reduction in the rate of ATP hydrolysis. This led to the hypothesis

that channel opening is affected by hydrolysis at NBDl while channel closure

is affected by hydrolysis at NBDZ.

Hwang et al. observed two types of channel gating, slow and fast, which

arose from activity at the two NBDs (Hwang et al., 1994). They observed that

single channels could be 'locked' open for prolonged periods following

activation by ATP by the addition of a non-hydrolysable analogue of ATP.

This is consistent with an inhibition of ATP hydrolysis at NBDZ, thereby

preventing channel closure. Prolonged bursts of channel activity were only

observed when the R domain was highly phosphorylated, emphasizing the

complexity of regulation by both the R domain and the NBDs.

4.1.2. Features of the CFTR Chloride Channel

4.1.2.1. Tlzree state mode1

To distinguish between open and closed states of CFTR, patch clamp

analysis is performed on single channels and channel conductance readings

are fil tered a t various frequencies. By heavily filtering conductance traces,

Gunderson and Kopito detectecl two open states of the CFTR channel, the

transition between the closed state (C) and the first open state (O,) is linked to

the binding of ATP to NBD2 (Gunderson and Kopito, 1995). 0, is a locked

open state, which is then transformed to the second open state (O,), by ATP

hydrolysis at NBD2. Channel closure is then spontaneous and likely results

from the dissociation of ADP from NBD2. Mutations in NBDl slow the

gating and result in reduced Po, however homologous NBD2 mutations

result in prolonged open times and prevent 0, to 0, transitions. These

results imply that ATP hydrolysis at NBDl is less important than at NBD2

and is possibly involved in switching CFTR to an active closed state, from

which NBD2 controls gating.

4.1.2.2. Doltble occupancy of the ion pore

The ion pore of CFTR is formed by the twelve transmembrane helices

of the two MSDs. There are several positively charged amino acids on the

inner surface of the pore, some of which are responsible for defining chloride

binding sites. Mutations leading to neutral substitutions at three arginine

residues may account for the pancreatic su fficient phenotype in CF patients

(R117H, R334W, and R347P) (Sheppard et al., 1993). Al1 three mutant proteins

are processed, but exhibit reduced single channel conductances. Furthermore,

R117H, wliich is located near the external end of the pore, is sensitive to

external pH, as channel activity is reduced when the histidine is uncharged.

By studying two different permeable ions, chloride and thiocyanate,

Tabcharani et al. determined that there exist two ion binding sites within the

(Tabcharani et al., 1993).

4.2. Regulation of Other Channels

Cells from CF patients have defective regulation of chloride

conductance. However, CFTR chloride conductance cannot account for the

complete electrophysiological defect seen in CF epithelia. There appears to be

an ou twardly rectified chloride channel (ORCC) whose activity is regula ted by

CFTR. Also, CFTR is involved in the regulation of sodium channels and acts

as an inhibitor of the rat epithelial sodium channel (rENa'C).

4.2.1. Outwardly-Rectifying Chloride Channels (ORCC)

Cells from CF patients, when compared to normal cells, appear to be

missing an outwardly rectifying chloride conductance of -40 pS. However,

several studies in which single purified CFTR channels have been

characterized, show that CFTR is a non-rectifying channel, with a

conductance of only 6-10 pS (Kartner et al., 1991; Welsh et al., 1992). However,

when recombinant CFTR is expressed in CF bronchial epithelial cells, not

only is the linear CFTR chloride conductance corrected, but PKA activation of

ORCC activity is also restored (Egan et al., 1992). This result suggests one of

two possibilities: either CFTR is regulating other channels, or CFTR itself is

assuming this ORCC function, by some undefined structural change.

Further evidence to support the mode1 that ORCCs are distinct proteins

and are regulated by CFTR came with the observation of ORCC in CFTR(-/-)

rrwuse nasal epirneliai ceils (batmer et al, ~ r r s ) . UKLL actlvlty 1s omervea in

the absence of CFTR only by prolonged (-400s) depolarisation of membrane

patches, but is not regulated by PKA. However, PKA regulation of ORCC

occurs . .. in normal (+/+) cells.

ORCC CO-purifies with CFTR from bovine tracheal epithelia as four

polypeptides (52, 85, 120, and 174 kDa) (Jovov et al., 1995). The 174 kDa

polypeptide corresponds to CFTR by reaction to CF antibodies. One or more

of the other three peptides is therefore providing ORCC activity. These CO-

purified proteins display chloride channel activity. The chloride channel

activity can be increased by the addition of PKA and ATP, but only on one

side of the bilayer. DIDS, an ORCC specific inhibitor, blocks ORCC activity on

the opposite side. ATP alone is not able to stimulate channel activity from

either side. Following immunodepletion of CFTR, the other proteins are still

phosphorylated by PKA.

Schwiebert et al. further characterized the relationship between CFTR

and ORCC and postulated that CFTR may mediate the transport of ATP out of

the cell, where the ATP can then stimulate proximal ORCC via a P,,

purinergic-dependant signalling mechanism (Schwiebert et al., 1995).

However, it is unclear how CFTR might facilitate ATP transport outside of

the cell.

4.2.2. ATP Conduc tance

Some evidence exists that CFTR may also be involved in the

conductance of ATP. External ATP is sufficient to activate non-rectifying

transfected with human CFTR (Cantiello et al., 1994). These findings are

consistent with the presence of a purinergic receptor signal transduction

mechanism whose activation by external ATP is linked to the activation of

CFTR in a CAMP-independent manner. As such, CFTR itself may be capable

of acting as a dual ATP and chloride channel. (Reisin et al., 1994).

Diphenylamine-2-carboxylate (DPC), a chloride channel blocker, is capable of

inhibiting the observed ATP conductance.

Conflicting data has been obtained by another group who were unable

to detect any ATP conductance in either intact organs, polarized human lung

ce11 lines, stably transfected mammalian ce11 lines, or planar lipid bilayers

(Reddy et al., 1996). This group argued that their observations were consistent

with the limitations imposed by the predicted pore diameter of CFTR (5.5A)

as being too small for ATP (IO&. They suggested that Cantiello et al. were

observing some combination of other ions passing through the CFTR

channel, or of ATP currents passing through alternate channels.

4.2.3. Sodium Channels

Airway epithelial cells bearing mutations in CFTR possess an increased

Na' conductance along with their well described defect of CAMP dependent C ï

conductance. CFTR is involved in the regulation of sodium channels,

although the mechanism of interaction is unclear. Xenopus oocytes, injected

with cDNA from al1 tliree subunits of the rat epithelial sodium channel

(rENa"C) and CFTR, express both amiloride sensitive sodium channels and

\ - - - - - - - - - - - - . - - - - - - . - . -., - - -- , ----- - -- ---- ----------

channels, as expected. An interaction between CFTR and rENa'C is

implicated because IBMX is capable of inhibi ting sodium conductance i n

oocytes injected with cRNA of CFTR-wt, but not with CFTR-AF508 (Mal1 et

al., 1996).

Stutts et al. also noticed that rENa'C expressed in Madin Darby caniné

kidney (MDCK) epithelial cells, generated large amiloride sensitive currents,

wliich were stirnulated by CAMP. However, co-expression with CFTR

resulted in smaller basal sodium currents that were inl-iibited by CAMP (Stutts

et al., 1995).

Regulation of sodium channels by CFTR is not limited to epithelial

cells in culture, expressing large levels of CFTR and rENa'C. Ling et al.

generated a CF phenotype in a renal amphibian epithelial ce11 line by

treatment with an antisense CFTR oligoiiucleotide (Ling et al., 1997). The

decreased expression of CFTR resulted in an increase in the Po of sodium

channels.

To test whetlier CFTR directly affects sodium channels, Ismailov et al.

co-incorporated into planar lipid bilayers, CFTR and rENa'C (Ismailov et al.,

1996). Tliey found tl-iat the single channel open probability of rENa'C was

decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP

activated CFTR, but did not have any effect on rENa'C. The presence of CFTR

also prohibited the inward rectification of the gating of tl-iis renal sodium

channel norinally induced by PKA-mediated pliosphorylation. The

whether or not CFTR-wt was conducting anions. However CFTR-G551D, a

non-conductive mutant, was not capable of sodium charnel regulation.

These results provide evidence that CFTR directly interacts with sodium

charnels, and that it must be functional to do so.

4.3. Regdafion of other ce11 functions: pH, sialylation, and plasma

membrane recycling.

CFTR also appears to be involved in the regulation of other ce11

functions, possibly via its effect on pH. CFTR is present and functional in

endosomes where it appears to regulate endosomal pH (Lukacs et al., 1992).

The absence of wild type CFTR, results in an increase in pH, which in turn

leads to secondary effects on plasma membrane recycling (Bradbury et al.,

1992) and sialylation of membrane glycoconjugates (Dosanjh et al., 1994).

Mutant CFTR also inhibits cytoplasmic acidification, a process important i n

apoptosis; cytoplasmic acidification results in the inefficient digestion of DNA

(Gottlieb and Dosanjh, 1996). The undigested DNA contributes to the high

viscosity of mucous in airways and other exocrine organs of CF patients,

contributing to the pathology of the disease.

V

5.1. CFTR Expression and Localisation

In-situ hybridization experiments have shown that CFTR is primarily

expressed in various epithelial tissues, including pancreatic ductal cells,

salivary glands, intestine, lung, and testis (Engelhardt et al., 1992; Sarkadi et

al., 1992; Trezise and Buchwald, 1991; Trezise et al., 1993; Trezise et al., 1992).

Mechanisms of up-regulation of CFTR expression have been explored.

Elevated levels of CAMP for prolonged periods (>8 h) result in an increase of

CFTR transcription.

and cholera toxin,

transcrip tional level

. .. Furthermore; various cAMP agonists, such as forskolin

are capable of increasing CFTR expression at the

(Breuer et al., 1992). The opposite effect is seen with

overexpression of protein kinase C (PKC). PKC expression is activated by

phorbol mysistate acetate (PMA) which causes a concomitant decrease i n

CFTR transcription. This reduction can be partially reversed by the addition

of PKC specific inhibitors (Breuer et al., 1993). The opposing effects of cAMP

and PKC may represent a regulatory pathway for CFTR dependant chloride

secretion. More recently, the complete genomic sequence of CFTR (from YAC

recombinant clones) has been introduced into the CHO genome. The YAC-

CFTR leads to the production of full length, functional human protein

(Mogayzel et al., 1997). The CHO cells provide a limited mode1 in that they

are not epithelial tissues, but will allow for further studies of the regulation of

CF gene expression.

mediating chloride transport. Apical localization has been shown by

immunocytochemical siudies using a number of different antibodies

(Denning et al., 1992; Kartner et al., 1992). This localisation is conserved with

the CFTR ortholog of the dogfish, where it has been found lining the lumen

of the rectal gland (Marshall et al., 1991). In addition, as much as 50% of

maturely glycosylated CFTR is also located in the intracellular vesicles where

it appears to be involved in regulating endosomal pH (Lukacs et al., 1992).

It is interesting to note that the epithelial cells lining the pulmonary

airways express relatively little CFTR. However, ion conductance in these

cells easily measured, and the absence of CFTR in the lung epithelium results

in the most serious pathophysiological consequences of CF (Welsh et al.,

1992).

5.2. Glycosylation

CFTR is glycosylated at two asparagine residues: NB96 and N900. Upon

progression of nascent CFTR along the trafficking pathway, the actions of

different glycosidases alter the oligosaccharide with modifications that can be

monitored by SDS-PAGE. The fully processed mature protein migrates as a

broad band at about 170 kDa (Band C), whereas CFTR that has been retained in

the ER appears as a sharp band at 140 kDa (Band B). Treatment with N-

glycanase (PNGase F), a glycosidase that cleaves al1 N-linked oligosaccharides,

leaves a single unglycosylated protein which migrates sligl~tly faster than the

- - - - -

been useful in monitoring the processing of ÇFTR. For instance, only about

25% of CFTR-wt becomes maturely glycosylated, and CFTR-AF508 reveals a

unique pattern in which only core glycosylation is seen, a finding consistent

with CFTR-AF508 being arrested in the ER. Other mutant CFTR proteins

appear to have distinct glycosylation patterns (Rommens, unpublished data).

These other CFTR derivations may be arrested in a specific post-ER

cornpartment, or may be slowed in efficiency of transport.

CFTR glycosylation can be used as a marker for processing, and may be

involved in interactions with extracellular proteins. However, it is not

necessary for the chloride channel activity of the protein. Various inhibitors

of oligosaccharide processing have had effects on the molecular weight of

CFTR, but they did not affect the CAMP-stimulated chloride secretion i n

epithelial cells, or localisation of CFTR to the apical membrane (Morris et al.,

1993). Also, mutations of the glycosylation sites had no effect on function or

localisation of the protein (Cheng et al., 1990; Gregory et al., 1991).

5.3. Association with Chaperones

CFTR glycosyla tion is important for its association with molecular

chaperones. Calnexin, an ER membrane chaperone, has been shown to

associate with both wild type and CFTR-AF508. In the case of CFTR-wt, some

of the protein is eventually released, as it is transported on to the Golgi.

J

degraded (Pind et al., 1994).

It has been well documented that proteins can be retained in the ER

until a desired folded state is achieved and release is facilitated. The

oligosaccharide molecule which is transferred onto al1 glycosylated proteins

possesses three terminal glucose residues. Before exiting the ER, the

outermost glucose residue is hydrolysed by glucosidase 1. The other two are

cleaved by the successive action of glucosidase II. However, UDF-

G1c:glycoprotein glucosyltransferase preferentially adds one glucose molecule

to the unglucosylated, high mannose glycans of unfolded protein. It is this

molecule, with one remaining glucose residue that is retained in the ER by

direct association with calnexin. In this mechanism of trimming and

reglucosylation, unfolded protein remains in the ER until it becomes either

properly folded or degraded (Hebert et al., 1995).

5.4. Poor Efficiency of Processing

As mentioned previously, only about 25% of CFTR-wt is maturely

glycosylated and delivered to the apical membrane (Marshall et al., 1994;

Ward and Kopito, 1994). The remaining CFTR-wt is degraded with kinetics

indistinguishable from CFTR-AF508 (t,,, = 33 min and 27 min, respectively).

In order to escape the ER degradation pathway, some CFTR-wt

undergoes a n ATP-dependant conformational maturation. The mutant

- - - L

physiological conditions, and therefore cannot exit the ER (Lukacs et al., 1994).

5.5. CFTR Degradation

The rapid degradation of both normal and mutant CFTR involves

multiple proteolytic systems (Jensen et al., 1995). One such system is the

Ubiquitin-Proteosome pathway in which protein is polyubiquitinated and

thus 'marked' for degradation. Ward et al. attempted to determine whether

the blockage of this pathway would enhance the efficiency of CFTR

processing, thereby possibly providing a therapy for CF processing mutations.

They discovered that although levels of immature CFTR could be maintained

for extended periods, there was no overall increase in levels of mature CFTR

when the ubuquitin-dependant protease pathway was blocked (Ward et al.,

1995).

6. CFTR Mutations

6.1. Classes of CFTR Mutations

There are more than 700 known mutations which are involved i n

cystic fibrosis (Tsui, 1994-1997). These have been loosely classified into 5

groups.

6.1.1. Defective Protein Production

The first group of mutations results in defective protein production

caused by nucleotide changes, insertions, or deletions that lead to premature

mutations.

6.1.2. Defective Protein Processing

The second class of mutations are categorized by defects in protein

processing. This class of mutations form the basis of most cases of CF. The

most common mutation, AF508, belongs to this group because it results in a

protein which is unable to mature properly and is consequently degraded i n

the ER (see section 6.2). There are also other mutations, several of which

occur in NBD1, which also have defective processing and consequently result

in reduced levels of functional protein at the plasma membrane. Many

homologous mutations placed in NBDP do not affect protein processing and

appear less detrimental to function. This indicates a higher susceptibility of

NBDl over NBD2 to the cells quality control mechanisms and suggests a lack

of equivalence of the two NBDs in function (see section 5.2; Gregory et al.,

1991).

6.1.3. Defective Regulation

The third class of mutations result in defective regulation of the

chloride channel. G551D belongs to this class because it appears to be

processed similar to wild type protein, yet does not appear to function

normally and results in a severe, pancreatic insufficient (PI), clinical

phenotype.

The fourth class of mutations results in a defect in the conductance

properties of the channel. Such mutations occur within the MSDs and alter

the ion pore (Sheppard et al., 1993; Tabcharani et al., 1993).

6.1.5. Alternative Splicing

A fifth class of mutations has also been suggested in which there may

be a reduced level of protein at the apical surface of the cell. Such mutations

could arise from unstable or inefficient splicing of mRNA.

6.2. AF508 is a Processing Mutation

By understanding the mechanisms by which CF mutations cause

disease, it is hoped that perhaps, treatments can be developed to bypass the

deficiency. In the case of the most common mutation, AF508, which accounts

for 68% of disease alleles, the mechanism of disease is a failure of CFTR to

achieve a maturely folded state and to be transported to the site of required

function (i.e. the apical membrane). Because of this inability to fold correctly,

CFTR-AF508 is retained in the ER by interactions with different chaperones,

until it is ultimately degraded. Consequently, immunoelectron microscopy

has located CFTR-AF508 only in the ER, whereas CFTR-wt is also located at

the plasma membrane (Pind et al., 1994; Yang et al., 1993).

However, when CFTR-AF508is expressed in Xenopus oocytes and Sf9

cells, the mutant protein is processed functional (Kartner et al., 1991; Li et al.,

1993; Ling et al., 1997; O'Riordan et al., 1995). Since these cell lines are

- ..

might be a temperature sensitive folding mutation.

Pulse chase experiments showed that when CFTR-AF508 is translated at

3TC, a maturely glycosylated form could be produced when processing was at

26'C (Denning et al., 1992). The addition of glycerol permits CFTR-AF508 to

achieve a maturely folded state (Sato et al., 1996), supporting that a folding

defect for the CFTR-AF508 is prevents correct protein processing.

Furtherrnore, when CFTR-AF508 is correctly processed, it is also

capable of producing chloride charnels with characteristics similar to CFTR-

wt, although reduced open probabilities are detected. In addition to being

moderately less conductive, CFTR-AF508 also has a reduced half-life of 4-7h

(cf. >24h for CFTR-wt) (Denning et al., 1992; Lukacs et al., 1993).

Revertants for this folding mutation were discovered upon the

introduction of STE6-CFTR chimeras into yeast, taking advantage of the role

of STE6 in mating (Teem et al., 1993). The H5 chirnera, consisting of STE6

with a homologous segment of CFTR-NBD1, resulted in a 12% mating

efficiency for NBD1-wt, but only 0.15% for the corresponding NBDLAF508.

Two mutants, R553M and R553Q, were able to restore mating efficiency when

present in cis with AF508. Introduction of these revertant mutations into

human CFTR partially corrected both the processing and chloride

conductance defects of CFTR-AF508. It is interesting to note that R553Q has

also been found to be associated in cis with AF508 in a patient with a mild case

of CF (Dork et al., 1991).

Defective intracellular transport is probably the mechanism of

dysfunction for a significant number of CF mutations. A number of studies

have shown that many mutations occurring in NBDl result in the absence of

maturely glycosylated CFTR, with concomitant lack of chloride channel

activity (Cheng et al., 1990; Gregory et al., 1991). Homologous mutations i n

NBD2 do not appear to have serious effects on processing, indicating that they

may be less susceptible to the cell's quality control mechanisms. Indeed, a

deletion mutation in which al1 of NBD2 is missing is still properly localized

to the plasma membrane, although it has no function (Rich et al., 1993b).

In other cases, where the protein is correctly processed, mutations in

NBDl are most likely involved in preventing ATP binding/hydrolysis or the

conformational changes associated with this activity.

G551D is a mutation associated with severe CF, although the protein

has been showed to be correctly processed (Gregory et al., 1991). The glycine at

position 551 is highly conserved across the ABC superfamily, and the parallel

mutation in STE6 (G509D) results in a drastic reduction in mating efficiency

(0.5%) (Berkower and Michaelis, 1991).

CF type mutations have also been generated in human P-glycoprotein

(Hoof et al., 1994). In this study, the severe NBDl mutations, AF508, S549R,

and G551D, were introduced into MDR1 and resulted in chemosensitization

to colchicine. Furthermore, the AF508 revertant mutation, R553Q (alone),

actually resulted in an increased resistance to colchicine.

- - -

suggest some structural and functional similarities. In particular, the GGQR

sequence (aa. 551-553) appears to form part of the nucleotide binding pocket

and may serve as a conformational ON/OFF switch which activates the

chloride channel (Manavalan et al., 1995).

Of particular interest are those mutations in NBDl which result in a

'mild' pancreatic sufficient phenotype. Based on phenotype, it would appear

that they are capable of 'partial' function. This could be due to reduced or

partially blocked processing, or through a mild effect on protein function.

Alternatively, their processing may be affected differently in different tissues.

Two such mutations, A455E and P574H, both result in protein with

normal conductive properties when studied by patch clamp analysis. The Po

of CFTR-P574H was higher than that of CFTR-wt because of prolonged bursts

in spite of long channels closures. However, the overall currents generated

by these mutants in cells is likely reduced because of reduced overall levels of

mature protein (Sheppard et al., 1995).

Very little CFTR-wt is required to prevent a classical CF phenotype;

only 2236 of normal CFTR transcript is required for normal lung function.

This could be determined from the 5T splice variant that is present in 5% of

the general population. This variant actually affects splicing to the extent that

it results in mRNA with a loss of exon 9 (and thus does not lead to functional

polypeptide), but shows variable expression between individuals and i n

different tissues within an individual (Rave-Harel et al., 1997). The presence

-- --------.- --. -- . . --- -, r- ----"-O' Y - ---LI*..*.- -*"YU-" * * L U I --r--.' -=

least in part, why PS mutations do not affect pancreatic tissue to the same

extent as they affect other tissues.

7. NBD Studies

A number of groups have generated peptides corresponding to part or

al1 of both NBDs.

A 67 amino acid synthetic peptide corresponding to amino acids 450-

516 (containing the Walker A motif of NBD1, but lacking Walker B) was

generated and found to be capable of binding to the ATP analogue, TNP-ATP.

Furthermore, this binding could be competed with ATP, although only at

significantly higher concentrations (Thomas et al., 1991). A similar 66 amino

acid peptide corresponding to the AF508 mutation was also capable of binding

ATP but with relatively reduced affinity. Perhaps more interesting was the

finding that AF508 mutation resulted in a reduction in B-structure, and

reduced stability upon denaturation with guanidinium chloride (Thomas et

al., 1992).

Chimeric proteins in which regions of NBDl or NBDP were fused to

glutathione-S-transferase (GST) or maltose binding protein (MBP) have been

generated by different groups, and have been expressed in E. coli. Upon

refolding under a variety of conditions, some groups were able to detect ATP

binding over a broad range (0.04 - 5 mM) (Hartman et al., 1992; Ko et al., 1993;

Logan et al., 1994; Randak et al., 1995). One group was also able to detect

Yike et al. generated a series of six different peptides, corresponding to

portions of NBD1, which were fused to a 6XHis tag for purification, then

refolded them under a number of buffer conditions. One peptide,

corresponding to amino acids 384-650, remained soluble following dialysis

into buffer containing polyethylene glycol (PEG 3350) and showed some

secondary structure by CD. The peptide was capable of binding to ATP,

however, it was completely excluded from a gel filtration column, indicating

that it was somehow forming soluble aggregates.

Very recently, the problem of aggregation was alleviated by refolding a

His-tagged NBDl peptide corresponding to amino acids 404-589 (exons 9-12) in

the presence of 0.4M L-arginine (Qu and Thomas, 1996). The guanido group

acts as a mild denaturant and so permits arginine to shield both ionic and

hydrophobic interactions through its mildly amphiphilic nature.

8. NBDl Expression, Purification and ATP Binding

Nucleotide binding domains are crucial components in many proteins.

Their essential roles have been highlighted in CFTR by the dire consequences

of mutations in these domains to CF patients. The cellular consequence is a

loss of a chloride conductance in the apical membrane of the epithelial cells

via channel malfunction or complete absence of protein. Experimental

results of the analysis of parallel mutations within the NBDs clearly point to

molecule. This is manifested at two levels: first, at the level of protein

trafficking and second, at the regulation of the gating of chloride conductance.

At the structural level, mutations that have been identified to affect

amino acids at or near the recognized motifs of NBDs; Walker A, Walker B,

or Walker C signatures must have immediate consequences that would

predict effects on local structure that alter nucleotide binding and/or

hydrolysis. These direct consequences will be most readily studied away from

the context of the entire multidomain protein.

In order to better understand the function of NBDl and how mutations

in NBDl can cause disease, 1 cloned a series of NBDl coding regions that differ

in length into various expression systems for peptide expression, purification,

refolding, and assessrnent for ability to bind ATP and ATP analogues. My

findings are consistent with previous and concurrent studies which show

that NBDl can only be refolded into soluble monomeric species under non-

physiological conditions. Fur thermore, these conditions are so severe as to

make functional evaluation of the NBDl peptides challenging.

Materials and Methods

1. NBD Expression Vectors

1.1. Cloning of NBD13,, into the pPINPOINT expression vector

CFTR cDNA frorn pBQ6.2 (Rommens et al., 1989) was restricted with

Fsp 1 and Eco RI to isolate the fragment coding for amino acids 348-699 of

CFTR, which correspond to al1 of the putative NBDl and a portion of the R

domain. (Another peptide corresponding to amino acids 348-605 was also

isolated by restriction of pBQ6.2 version 2 with Fsp 1 and Bst EII.) These

fragments were treated with T4 DNA Polymerase then gel purified. The

inserts were then ligated into the pPINPOINT vector that had been restricted

with Eco RV, and the ligation mix was used to transform DH5a FfIQ

competent cells (Gibco). Cells were plated ont0 LB agar with 50 pg/ml

ampicillin, 10 pg/ml kanamycin, and 0.2% glucose.

1.2. Cloning of NBDl,,, NBDLWCW and NBDl/R,,,,,, into the

PET expression vector

Cloning into the PET expression system was performed by shuttling the

fragments encoding the PINBDl fusions, including most of the biotinylated

peptide, into the PET vector to include a 6XHis tag in frame at the 3' (C-

terminus) end. In order to do this it was necessary to remove a large region of

- - - -

stranded oligonucleotide, containing the desired restriction sites.

BiopET-top 5' C ATG GAG AAA AAA GTC GAC GGA TCC GCG GCC GCC 3 '

BiopET-bottom 3 ' CTC TTT TTT CAG CTG CCT AGG CGC CGG CGG AGC T 5 '

Figure 2. Sequence of Synthetic Oligonucleotide Insert. The two synthetic oligonucleotides, BiopET-top and BiopET-bottom were ameaied as described (section 1.2) and ligated into pET28 vector.

Two synthetic oligonucleotides, BiopET-top and BiopET-bottom

(Figure 2)' were dissolved in Tris, 10 mM: EDTA, 1 mM (T:E) at a

concentration of 1-1.5 @pl. The two oligonucleotides were then mixed and

heated to 7Q°C, then allowed to anneal by cooling to 4'C over 2 hours. The

annealed oligonucleotides were then ligated into pET28b, that had been

restricted with Nco 1 and Xho 1 (New England Biolabs), and gel purified.

NBDl,,,, and NBDl,,,-,, were shuttled into pET 28M by directional

cloning, using the Sa1 1 and Not I restriction sites flanking NBDl (including

most of the biotinylated peptide).

NDB1/R3,,, was cloned by PCR amplification of the R domain,

ligatied into pETNBDl,,,,,. The PCR product was verified for accuracy by

dideoxy sequencing.

expression vector

PCR primers were designed to amplify the indicated NBDl cDNA with

an N-terminal start codon and a C-terminal 6X His tag. Nco I and Not 1

restriction sites were placed at the 5' and 3' ends of the PCR product to allow

for directional cloning into the pET28 plasmid vector. The sequence of the

PCR primers for each peptide is indicated in Table 1. PCR was performed in a

DNA Thermal Cycler (Perkin Elmer, Mode1 480), with the following

conditions: 20-30 cycles, 1 min denaturation Q 94'C, 1 min annealing @i 5B°C,

and 2 min elongation Q 72'C. The PCR reactions contained 10 ng of plasmid

that had been linearized with Eco RI, 200 ng of each primer, 200 mM dNTPs,

and 2 units of Vent polymerase in thermopol buffer (NEB). The PCR

products were then restricted with Nco I and Nof I and ligated into the Nco 1

and Not 1 sites of the pET28 vector. Al1 PCR generated inserts were verified

for accuracy by dideoxy sequencing across the amplified region.

L a V A G 1.. b&\ L a a I L 6 L L a I V & A I L P U A L CYLIUZ~- L A N G ~ V U V V V Y ~ ~ A -r. y a u r r b r w . v b & - UY-u AYA

amplification and doning of NBDl peptides into PET 28. The text in bold represents the first codon of the corres~ondhg peptide. - A

NBDl Peptide (Primer name) NBD'404-589 (N404C)

Synthetic Oligonucleotide Primer Sequence

M G F G E L F E CGC.GCC.ATG.GGA.TTT.GGG.GAA.TTA.TTT.GAG

M E Y N L T T T CGC.GCC.ATG.GAA.TAT.AACCTTAAACGGACT.ACAAG

GCT.CTC.GAG.AGA.ATC.ACA.TCC.CAT.GAG

M A N N N N R K T S N C G C . G C C . A T G . G C A . A A C . A A T . A A C . A A T . A G A . A A A

1.4. Construction of Vectors Encoding NBDl peptides

Cysteine residues at positions 491 and 524 of NBDl were each mutated

to alanine and to serine. Site-directed mutagenesis was performed by PCR

amplification using mismatched primers and wild type DNA template.

Primers were designed across the codon to be mutated for both coding and

non-coding strands, such that there was 22 base pairs of complementary

sequence* in the corresponding primers (see Table 2). The mutant primers

were then separately used with the NBDl peptide primer (from Table 1) of the

opposite strand to amplify NBDl cDNA. This resulted in PCR products

corresponding to each half of the NBDl cDNA, with 22 base pairs of

overlapping sequence, flanking the mutated codon. These two PCR products

VVCLC L L ~ C A L ~VLJICU UALU UDGU a3 LCILL~IULG IIL LILL I LI\ UIAL ~ I A I A L U LAVAL VA A v Y Y A

cDNA using the NBDl PCR primers (Table 1).

Table 2. PCR Primers for C491AlS and C524MS NBDl Mutations.

Primer Synthetic Oligonucleotide Primer Sequence

C491A/S-c 5 ' A ATT TCA TTC (T/G)CT TCT CAG TTT TCC TGG A 3 '

C491A/S-n 3 ' A CCT TCT TAA AGT AAG (AIC) GA AGA GTC AA.A5'

C524A/ S-c 5 ' c ATC AAA GCA (T/G) CC CAA CTA GM GAG GAC 3 '

C524A/ S-n 3 ' CT TCG CAG TAG TTT CGT (A/C)GG GTT GAT CTT 5 '

2. Expression, purification, and refolding of NBDl

peptides

2.1. Expression, purification, and refolding of NBDl,,, expressed

from pPINPOINT Vectors

1 L of LB broth with 100 pg/ml Ampicillin, 10 pg/ml of Kanamycin,

0.2% glucose, and 2 PM Biotin was inoculated with 10 ml of overnight culture

(DH5a F'IQ). The culture was incubated on a rotary shaker (200 rpm) at 37'C

until the OD,,, was -0.5 (0.4-0.6). Isopropyl-P-D-thiogalactoside (IPTG) was

added to a final concentration of 100 p M and the culture was incubated for

another 3 h. The cultures were centrifuged at 5000g for 15 min, the

supernatant was decanted, and the cells were resuspended in 10 ml of lysis

buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 8.0). Lysozyme was added

to a final concentration of 1 mg/ml and the suspension was rocked at 4'C for

suspension was rocked for another 5 min. The lysed ce11 suspensions were

then sonicated with a Misonix ultrasonicator to shear DNA and thereby

reduce the viscosity of the suspension. The inclusion bodies were then

collected by centrifugation at 12000g for 15 min in a Beckman J-2M centrifuge

with a JA-20 rotor. The supernatant was decanted, and the inclusion bodies

were resolubilized in 8 M urea, and proteins refolded by stepwise dialysis (1 M

decrements of urea) into stabilising buffer (50 rnM Tris, 100 mM NaCl, 6 rnM

MgCl,, 1 mM EDTA, 1 mM DTT, and 5% glycerol, pH 8.0).

2.2. Expression, purification, and refolding of NBDl,,,,, Expressed

from PET Vectors

100 ml of LB broth with 30 pg/ml of Kanamycin was inoculated with 1

ml of overnight culture (BL21), then incubated on a rotary shaker (200 rpm) at

37'C until the OD,, was -0.5 (0.4-0.6). IPTG was added to a final

concentration of 100 pM and the culture was incubated for another 3 h. The

culture was centrifuged at 5000g for 15 min, the supernatant was decanted,

and the cells were resuspended T:E buffer. Lysozyme was added to a final

concentration of 1 mg/ml and the suspension was rocked at 4'C for 20 min.

Triton X-100 was added to a final concentration of 0.1% and the suspension

was rocked for another 5 min. The lysed ce11 suspension was then sonicated

and the inclusion bodies were collected by centrifugation. The supernatant

\vas decanted, and the inclusion bodies were waslzed with 10 ml of T:E buffer

- - binding buffer (8M urea, 100 mM Tris, p H 8.0, 1 N NaCl 5 mM imidazole),

filtered through a 0.45 Pm filter, and applied ont0 2.5 ml of charged His-Bind

(Novagen) Nickel affinity resin. The resin was washed with 10 ml of binding

buffer with 50 mM irnidazole. The protein was then eluted with 6 ml of

binding buffer with 400 mM imidazole. The eluted sample was then

concentrated to a final concentration of 1 mg/ml in a centricon-30 (Amicon)

ultrafiltration concentrator. The protein was refolded by 10 fold rapid

dilution into various buffer conditions at 4"C, as indicated in Table 3.

Table 3. Refolding Conditions for pETNBDl,,,,. Protein was refolded by 10 fold rapid dilution into the buffers indicated. * With buffers D and E, the protein was initially refolded into buffer with 2 mM Glycine, pH 10, incubated for 2 h at RT, and finally neutralized with 20 rnM Tris, pH 6.8.

Buffer A B C D * E * Tris, pH 7.2 20 mM 20 mM 20 m M

Glycine, p H 10 2 mM 2 mM

Tris pH 6.8 20 mM 20 mM

NaCl 200 mM 200 mM 200 mM 200 mM 200 mM

MgCl, 10 mM 10 mM 10 mM 10 mM 10 mM DTT 1 mM 1 mM 1 mM 1 mM 1 mM

Triton X-100 0.1% 0.1%

Tween-80 0.1%

Expression and isolation of NBDl inclusion bodies in E. coli BL21 was

carried out as described (section 2.2). The inclusion bodies were dissolved in 5

ml of 6 M guanidinium chloride (GdmCl), 100 mM Tris, pH 8.0, filtered

through a 0.45 p filter, and applied onto 2.5 ml of charged His-Bind

(Novagen) Nickel affinity resin. The resin was washed with 10 ml of 6 M

GdmCl, 100 mM Tris, pH 8.0. The protein was eluted with 5 ml of 6 M

GdmC1, 100 mM HOAc, pH 4.5, then precipitated by dialysis against dH20.

The precipitated protein was dissolved in 500 pl of 8 M GdmCl and 40

pl of 1.5 M Tris, pH 8.8 and 60 pl of 1 M DTT was added. The suspension was

incubated at room temperature overnight, then dialysed against 20 mM MES

pH 6.0. The precipitate was washed five times with 20 mM MES pH 6.0 to

remove any residual DTT. The purified, reduced protein was dissolved in 6

M GdmC1, 20 mM MES, 1 mM EDTA (pH 6.0) and the concentration was

determined spectrophotometrically using a calculated extinction coefficient

(Gill and vonHippel, 1989) of 13,490 M-'crn-' for the NBD14,4-,,, peptide, 22,900

M-lcm-' for the NBDl,,,,,, peptide, and 15,930 M-'cm-' for the NBDI,,,,

peptide. Peptides were refolded by rapid dilution into buffer containing 400

mM L-Arginine, 2 mM EDTA and 100 mM Tris pH 8.0 (or pH 7.0, or MES pH

6.0).

-

I I

Peptides were refolded as described above. They were

with the indicated concentrations of Guanidinium chloride

temperature. Intrinsic tryptophan fluorescence was rneasured

then incubated

for 2h at room

by excitation at

280 nrn and reading emission at 335 nm. The data were fit to the equation:

(rnlm[Gdrn~l]-~~/R~) e

F = (ub - lb)

l + e (rn I-[GdmCl]-AGRT) + lb

where:

F = fluorescence ml = slope of 1nK vs. [GdmCl] curve; K=[folded] /[unfoldedl [GdmCl] = concentration of denaturant AG = Gibb's free energy R = universal gas constant T = temperature in degrees Kelvin ub = upper baseline lb = lower baseline

2.5. Light Scattering of Refolded Proteins.

As a means of monitoring the aggregation of NBDl peptides, light

scattering was measured upon rapid dilution into refolding buffer. Light

scattering was measured on a Hitachi F-2000 fluorimeter with excitation and

emission wavelengths set at 400 nm. The scattered light was measured at a

90' angle from the incident beam, and monitored over time.

200 pl of refolded NBDl (1.5 PM) was loaded ont0 a Superose 12

(Pharmacia) gel filtration column equilibrated with 400 mM L-Arginine, 150

mM NaC1, 20 mM Tris pH 8.0 (or pH 7.0 or 20 mM MES, pH 6.0). The flow

rate of 0.5 rnl/min was controlled by a Pharmacia FPLC system with

spectrophotometric detector at 280 nm, and recorded as a tracing on a n

analogue chart recorder.

3. ATP binding assays

. .

3.1. 8-azido-ATP binding

Azido-ATP binding was performed at 4'C in the dark. 8-azido-ATP ([a-

"Pl or [y-32P]) (5-10pM final concentration) was added to 10pl protein sample,

either in an amber eppendorf tube or in the well of a Coors white porcelain

spot plate. The volume was brought to 20pl with dH,O, and the solutions

were incubated for 5 min. The solutions were irradiated for 5 min with U V

light from an inverted light box directly above the samples. Any unreacted 8-

azido-ATP was quenched with 5 pl of 5X SDS-PAGE loading buffer (1 M Tris,

pH 6.8,100 mM DTT, 10% SDS, 50% glycerol, and 0.01% bromphenol blue).

The proteins were analysed by SDS-PAGE on a 12% polyacrylamide gel,

stained with Coomassie Brilliant Blue (R-250) and dried ont0 filter paper.

Radioactivity was quantitated by exposure on autoradiographic film (Kodak

software).

3.2. Intrinsic Tryptophan Fluorescence

Refolded peptide was incubated with varying concentrations of ATP.

Tryptophan fluorescence was then detected by excitation at 295 nm (10 m m

slit width) and measuring emission at 335 nm (10 mm slit width).

Conformational changes associated with ATP binding are sometimes reflected

by changes in intrinsic tryptophan fluorescence. Quantitation of binding was

performed using a graphical procedure, correcting for imer filter effect

(Mertens and Kagi, 1979).

3.3. TNP-ATP

Fluorescence enhancement of trinitrophenyl-ATP (TNP-ATP,

Molecular Probes) was used to assay for ATP binding to NBDl peptides.

Varying concentrations of TNP-ATP were incubated with peptide and excited

at 408 nm (10 mm slit width); emission was measured at 538 nm (10 mm slit

width) using a Hitachi F-2000 spectrofluorimeter with sample chamber

maintained at 25'C. Fluorescence enhancement was calculated by subtraction

of the fluorescence measurements of TNP-ATP alone and peptide alone from

the measurements of TNP-ATP and peptide together.

Results and Discussion

In order to directly study the effects of mutations in NBDl on ATP

binding, 1 expressed and purified NBDl peptides separate from NBD2 and the

rest of CFTR (Figure 3). Sequence alignment, performed upon cloning of the

gene (Riordan et al., 1989), assigned amino acids 433-585 as representing the

domain. The domain included exons 9 through 12 and was largely based on

alignment to other ABC transporters. However, it was unclear whether this

assignment accurately represented the domain within the multidomain

polypeptide. Therefore, in order to ensure that al1 required elements were

present, 1 initially chose a peptide possessing arnino acids 348-699. This

peptide corresponded to the last few amino acids of the sixth transmembrane

a-helix, al1 of NBDl and a portion of the R domain. Although it was

recognized at the time that the boundaries of this peptide might later need to

be changed, it appeared to be a reasonable starting point. NBDl,,,,, was

expressed in the PINPOINT system (Promega) which fuses a 13 kDa bacterial

peptide to the N-terminus of the target protein. Biotinylation occurs at a

lysine residue in this peptide and is carried out by the native biotinylating

enzymes of the host ce11 upon recognition of the biotinylation sequence. This

biotinylated peptide allowed for the use of streptavidin-horseradish

peroxidase (Streptavidin-HRP) for detection by enhanced cherniluminescence

(ECL). Furthermore, the protein could be purified on streptavidin agarose

after expression.

Figure 3. NBD1 Constructs. The boundaries shown correspond to those based ai alignment to ABC proteins (Riordan et al., 1989).

I S b /

1.1. Expression and Purification of PINBDl Peptides

biotinylated peptide

PINBDl,,,,, expression in E. coli is illustrated in Figure 4. An expected

0 1 I

PINBD1348699

55.6 kDa biotinylated protein was expressed after induction with IPTG. The

Western blot shown in Figure 4 also shows the presence of two native

1 . , a , , I I - /

) > ) , 1, f. . q < ," - 1 '

PINBDI biotinylated peptide 1 , , / ,

I , r '

, ;.

biotinylated proteins present in al1 lysates.

biotinylated peptide P ~ ~ ~ M I I - ~ ! B (H&, ,

~44444./4444 1 , - , . . a t I

1 , L ' a a , * , , , a , J

biotinylated peptide ~ ~ ~ ~ 3 4 8 . 8 5 8

Figure 4. Expression of pPINBD1-wt. NBDl peptide corresponding to amino acids 348- 699 of CFTR was expressed as a fusion protein to a 13 kDa peptide, which gets biotinylated in E. coli. Expression was under the control of the tac promoter and induced using 100 pM IPTG. The left panel shows the sodium dodecylsulphate - polyacrylamide gel electrophoresis (SDS- PAGE, 12%) gel, stained with Coomassie Brilliant Blue (R-250), and the right panel shows the Western blot, incubated with a Streptavidin-Horse Radish Peroxidase (Streptavidin-HRP) conjugated protein, and developed by enhanced chemiIuminescence (ECL, Amersham). Lanes 1 and 2 are whole cell lysates of DH5a transformed with the pPINPOINT vector alone, prior to and after induction, respectively. Lanes 3 and 4 are whole ce11 lysates of E. coli. DHSa transfonned with the pPINBD1-wt vector coding for the fusion with NBDl,,,,, prior to Euid af ter induction, respectively.

The NBDl fusion protein was found in the insoluble fraction of lysed

cells, indicating that it was forming inclusion bodies. Although it is preferable

to obtain soluble protein from the supernatant of lysed bacteria, the formation

of inclusion bodies is often a result of the overexpression of a heterologous

protein in E. coli . Proteins which form inclusion bodies must be refolded

into their native state. However, the conditions required for refolding are

different for different proteins and must be empirically determined.

1 attempted to retain NBDl in the soluble fraction by reducing the

temperature at which cultures were grownto 30°C and using lower

did not increase protein solubility. Therefore, 1 isolated and resolubilized the

inclusion bodies and determined refolding conditions for the NBDl protein.

Following lysis of DH5a expressing PINBD1,,,6gg, the inclusion bodies

were resolubilized in 8 M urea and refolded by dialysis into stabilising buffer

(see Methods, section 2.2). Dialysis was carried out both rapidly (directly into

stabilising buffer) and gradually (in 1 M decrements of urea). However i n

both cases, the resultant protein solution was cloudy, indicating that the

protein was aggregating upon removal of denaturant. In addition, the

purification of PINBDI,,,, was problematic and failed to bind to modified

streptavidin agarose (Promega). Consequently, subsequent experiments were

performed using only the partially purified protein, that had been isolated as

inclusion bodies. In addition to the PINBDl,,,,-wt protein, the two

mutants, PINBD134,6g9-A455E and PINBDl,,,-G551D were also partially

purified (Figure 5).

Figure 5. Partially Purified PINBDl proteins. The left panel is a 12% SDS-PAGE gel of PINBDl proteins stained with Coomassie Brilliant Blue. The right panel is a western blot of a similar gel, incubated with Streptavidin-HRP, and developed by ECL. The wt, A455E, and G551D fusion pro teins are represented in lanes 1, 2, and 3, respec tively . Lanes 4 and 5 axe BSA standards (25 and 50 pg/ml, respectively) for estimation of the approximate concentrations of the PINBDl proteins.

1.2. ATP Binding of PINBDl Peptides

Preliminary 8-azido-ATP binding experiments were performed on

crude ce11 extract of E. coli DH5a expressing PINBDl,,,,,, in stabilising buffer.

As can be seen in Figure 6, a number of proteins were labelled by the ATP

analogue, including one protein that corresponded to the biotinylated peptide

expressed in DH5a carrying the pPINBD1,,69, plasmid, that was absent in

cells containing the control plasmid. This observation was encouraging and

suggested that at least some of the expressed protein was capable of binding

ATP. However, because of the high levels of labelling to other proteins, it

was clear that ATP binding experirnents would need to be performed on

mutant variants.

Figure 6. [~x-~*P1-8-azido-ATP Binding to PINBDl Crude Extract. [~x-~*P]-8-azido-ATP (10 ph4) was incubated with lysed E. coli. DH5a cells expressing PINPOINT vector alone (lane 1) or PINBDl (lane 2) in amber eppendorf tubes. The samples were irradiated for 10 min w ith UV light, quenched with 5X SDS-PAGE loading buffer, then analysed by 12% SDS-PAGE. The proteins were transferred to a nitrocellulose support, and exposed on autoradiographic film (left panel). The membrane was then incubated with Streptavidin-HRP, and developed by ECL (right panel). The arrow indicates the position of the 55.6 kDa biotinylated PINBDl fusion peptide.

Because of the problems with purification of PINBDl,,-,, peptides,

ATP binding experiments could only be performed on partially purified

proteins. ATP binding experiments of PINBD1,,,~,g,-wt, -A455E, and -G551D

peptides using the û-azido-ATP analogue is shown in Figure 7. As depicted

in the Western blot, al1 three peptides appear to be equally abundant.

However, the labelling of PINBD1,,,-69g-A455E is markedly reduced compared

This result was consistent with the hypothesis that the inability of NBDl to

bind ATP was correlated to disease severity, and encouraged further

experiments to quantitate ATP binding. However, because of the relatively

low expression and poor purification seen with the pPinpoint system, it was

decided to switch to the PET expression system, in which protein expression

would likely be much higher, and the purification could be ~erformed under

denaturing conditions.

Figure 7. [a-32P]-8-azido-ATP Binding to Partially Purified PINBDl Fusion Peptides. [a-"q-8-azido-ATP (10 pM) was incubated with PINBDl proteins in amber eppendorf tubes. The sarnples were irradiated for 10 min with UV light, quenched with 5X SDS-PAGE loading buffer, tlien analysed by lZO/O SDS-PAGE. The proteins were transferred to a nitrocelluiose support, and exposed on autoradiographic film (left panel). The membrane was then incubated with Streptavidin-HRP, and developed by ECL (right panel). The arrow indicates the position of the 55.6 kDa biotinylated PINBDl fusion peptide.

2.1. Expression and Purification of pETNBDl,,,,

The expression of NBDI,,,,,, in the pET expression system (Novagen)

was greatly improved over that of pPINPOINT, typically yielding greater than

5 mg of protein from 100 ml of bacterial culture. In addition, since the protein

was engineered to include a 6X His tag at the carboxyl terminus, it allowed for

purification under denaturing conditions. Expression of pETNBDl,,,, is

shown in Figure 8. Although the biotinylated peptide was not needed for

detection of the NBDl peptide, it was maintained as a fusion to NBDl to

improve protein solubility and to provide a convenient way to compare

concentrations of different peptides.

M l 2 3 4 1 2 3 4

Figure 8. Expression of pETNBDl,,,,,,. The left panel is an SDS-PAGE gel of pETNBDl,,.,, proteins stained with Coomassie Brilliant Blue. The right panel is a western blot of a similar gel, incubated with Streptavidin-Hm, and developed by ECL. Lanes 1 and 2 are bacterial lysates of E. coli BL21 carrying the pETNBDl,,,, plasmid, before and after induction, respectively. Lanes 3 and 4 are the supernatant and pellet after lysis, respectively.

- - -- J

chromatography on His Bind (Novagen) resin, in the presence of 8M urea.

This resulted in >95% purity of protein preparations. Figure 9 illustrates

purification profile of pETNBD1,,,,gg in 8M urea. Further, the purification is

very efficient as al1 of the protein is eluted in the presence of 400 m M

imidazole, which cornpetes for Nickel ligands. Upon stripping of the nickel

from the column with 100 mM EDTA, it was noted that there was very little

protein remaining bound to the column after elution. Elution can also be

performed by reducing the pH to 4.5, thereby deprotonating histidine residues

in the His tag.

I i Figure 9. Purification of pETNBDl,,,4,,. Inclusion bodies were resolubilized in 10 ml of

binding buffer and applied to 2.5 ml of His-Bind resin (lane 1). The flow through was collected and passed over the resin three times (lane 2). The resin was then washed with 10 ml of binding buffer with 50 rnM Imidazole (lane 3) and the protein was eluted in 6 ml of binding buffer with 400 mM Imidazole (lane 4). The resin was the stripped with 5 ml of 100 rnM EDTA in 8 M urea (lane 5). Samples were analysed by SDS-PAGE (12%), and the gel was stained with Coomassie Brilliant Blue.

pETNBDI,,,, was refolded by both dialysis and rapid dilution

methods. Diaiysis of denatured protein to remove denaturant typically

resulted in precipitation of protein. However, rapid dilution under buffer

conditions containing the detergents Triton X-100, or Tween-80 (see Methods,

Table 3) resulted in protein that remained soluble for 12-20 hours. These

refolded protein solutions were used in subsequent ATP binding

experiments.

2.3. pETNBDl,,, does not bind ATP by either 8-azido-ATP or TNP-

ATP binding assays.

ATP binding experiments were performed on pETNBDl,,, using two

of the assays described (Methods section 3). It was found that there was no

enhancement in fluorescence of TNP-ATP upon incubation with

PETNBD~,,~~, that had been refolded as described (Methods section 2.2)

compared to denatured pETNBD1,-, (in 6M urea, not shown).

Furthermore, a high degree of background fluorescence was observed in the

presence of detergent.

Conversely, 8-azido-ATP was capable of labelling pETNBDI,,,,, as was

seen with PINBD1,4,,,. It was however noted that the labelling appeared to

be significantly weaker than was expected, possibly indicating that the protein,

although soluble, may have been aggregated. Such aggregation would only

allow for molecules that were located on the surface of the aggregate to be

- - with ATP, suggesting that the binding was non-specific.

3. Other Attempts to Find a Soluble NBDl Peptide.

As mentioned earlier, the domain boundaries of NBDl had not been

clearly defined. As a result, it was possible that the NBDl,-,, constructs were

either lacking a crucial region of NBD1, or alternatively, possessed an

unstable portion of an adjacent domain. In such events, it would be difficult

to obtain soluble and monomeric protein. In order to address this possibility,

two additional NBDl peptides were fused to the biotinylated peptide in the

PET expression system (see Figure 3). One peptide was shortened at the

carboxyl terminus to eliminate the portion corresponding to the R domain

(pETNBDlS,.,,,), and the other peptide was lengthened to possess NBDl and

al1 of the putative R domain (PETNBD~/&~,,,).

3.1. Expression of pETNBD1S ,.,,, The shorter NBDl peptide, corresponding to amino acids 348-605, was

expressed in the PET expression system and resulted in comparable

expression to what was seen with PETNBD~,,,~~. However, it did not appear

to be any more soluble than pETNBDl,,, nor was it able to specifically bind

ATP.

I - - - r - - - p - -- - -J' iD-OJD

The long NBDl peptide, corresponding to amino acids 348-858, as seen

in Figure 10, was poorly expressed and notably degraded. Consequently, it was

not possible to perform ATP binding experiments on this peptide. That the

instability of NBDl peptides becomes increasingly worse, and more prone to

degradation, as more of the R domain is included in the construct has also

been reported by others (Yike et al., 1996). Such degradation of peptides may

reflect a combination of problems. One problem could be that the metabolic

stress of producing such a large protein affects the health of the host bacteria

to result in less overall protein synthesis.

As seen in Figures 4 and 8, there were no detectable biotinylated

degradation products from the shorter NBDl peptides. Such a situation could

possibly arise if the protein was so insoluble that it immediately precipitated

and formed inclusion bodies after being synthesized. This would effectively

make the protein unavailable to bacterial proteases. However, if a protein,

such as the longer construct, remained soluble for longer periods of time, it

would conceivably be more susceptible to degradation.

Figure 10. Expression of pETNBD11 R,,,.,,. 12% SDS PAGE gel of pETNBDl/ R,,,.,,. The left and right panels represent the coomassie stained gel <and the Western transfer, respectively. Whole lysates of E. coli BL21, expressing pETNBDl/R,,,, from before (BI) and 2 h after (AI) induction with 100pM IPTG, were isolated. Induced ceils were also lysed and centrifuged at 2000g for 15 min. The pellet (P2000g) was isolated and the supematant was decanted and centrifuged at 12000g for 15 min. Both pellet (P12000g) and supematant (S12000g) were also analysed. (Note: growth and induction of pETNBDl/ R,, was perfonned at 30°C to minimize degradation.)

4. N B D 1404.~9, NBDl,,,,,, and NBD1, ,.,, Peptides

It appeared that the main difficulty in obtaining a soluble, monomeric,

and functional NBDl peptide was a result of not knowing the correct domain

boundaries. Furthermore, the peptides being used to date also comprised a 13

kDa biotinylated peptide which could possibly be interfering with function

and even solubility (despite Our hopes for the opposite effect). It was

therefore decided to pursue an NBDl peptide that not only possessed the

correct domain boundaries, but that also eliminated any extraneous amino

acid residues. Because of its high expression and ease in purification, the PET

system was used with some modifications.

- - -

sequences possible. These were effectively a methionine residue at the N-

terminus to allow for+a start codon, and eight amino acids at the C-terminus

including the six histidines of the His tag. Three separate peptides

corresponding to amino acids 404-589,384-649, and 415-675 were prepared (see

Figure 3). The boundaries of these peptides were chosen based on different

criteria.

The first peptide, NBD14,,,, was chosen based on work by Qu and

Thomas, where they were able to obtain soluble monomeric peptide by

refolding by rapid dilution into a buffer containing 0.4 M L-arginine. They

chose these boundaries based on the coding regions of exons 9-12, and

expressed this peptide in the PET expression system using the available

restriction sites (Qu and Thomas, 1996). While such a criteria was not

necessarily without weaknesses, it seemed a reasonable peptide to test and its

boundaries shared some proximity to the boundaries originally assigned by

sequence alignment to other ABC transporters (Riordan et al., 1989). Qu and

Thomas obtained a fusion protein with a 6X His tag and 18 extraneous amino

acids (coded for by the remainder of the vector's multiple cloning site) at the

N-terminus. Conversely, the NBDl,,,,,, peptide prepared in Our lab was

constructed with the 6X His tag at the C-terminus, and eliminated the

extraneous arnino acids of the vector.

NBDl,,,,,, was chosen based on a study of six peptides of different

lengths (Yike et al., 1996), in which it was the only one to remain soluble and

was different from that of Qu and Thomas in that the protein was refolded by

dialysis overnight into a buffer containing 0.1 g/l of polyethylene glycol (MW

3350). Although the protein appeared to be soluble, gel filtration revealed

that it was in fact excluded from the column, indicating the formation of large

aggregates (greater than ten protein molecules in size). Despite this problem

with aggregation, this peptide appeared promising because of its improved

solubility over the other peptides studied. We therefore chose to use this

protein, but to attempt to refold it using the protocol developed by Qu and

Thomas.

Finally, NBDl,,,,, was chosen based on two criteria: the amino

terminus was chosen based on molecular modelling using the structure of

Aspartyl Amino Transferase (F. Hoedemaker, D. Rose, A. Davidson, in press)

and the carboxyl terminus was chosen to include the conserved region of the

R domain (RDZ) (Dulhanty and Riordan, 1994a). Refolding was carried out in

the presence of L-arginine, which is believed to shield non-specific

hydrophobic interactions, thereby preventing aggregation.

Figure Il. Expression and Purification of NBDI,,.,,, and NBDl,,.,,. 12% SDS PAGE gel of NBDl peptides. Whole lysates of E. coli BL21, expressing pETNBDl,,.589 (Ieft panel) and pETNBDl,,-,, (right panel), from before (BI) and 2 h after (AI) induction with 100pM IPTG, were isolated. Induced cells were also lysed and centrifuged at 12000g for 15 min. Both pellet (P12000g) <and supernatant (S12000g) were analysed. The pellet was dissolved in 6M GdmCl, 100mM Tris, pH 8.0, and loaded ont0 a charged, equilibrated His-Bind column. The flow through (FT) was collected and passed over the column three times. The column was washed with 10 ml of 6 M GdmC1, pH 8.0, and 6 M GdmCl, pH 6.5, (both 1CKhnM Tris, Wash), then the protein was eluted with 5 ml of 6 M GdmC1,lOO mM HOAc, pH 4.5 (Elute). The column was then stripped of Nickel with 6 M GdmC1,lOO rnM EDTA (Strip).

Al1 three NBDl peptides were highly expressed in E. coli BL21, and

purified under denaturing conditions. The expression of NBDl,,-,, and

NBDl,,,,, are shown in Figure 11. As with the previous PET expressed

proteins (with the exception of pETNBDl/R,,,,) there was high level

expression following induction with IPTG. The proteins were exclusively

found in the inclusion bodies, and as such, were highly purified by

centrifugation alone following bacterial lysis. After resolubilizing the

proteins in 6 M GdmCl and purifying them over the His-Bind column, >95%

purification was achieved. The level of expression of NBDl,,,,, was

4.2. NBDl Peptides Remain Soluble and Monomeric in the Presence

of 0.4 M L-Arginine

The three NBDl peptides were refolded by rapid dilution in the

presence of 0.4 M L-arginine where they remained soluble. They were then

passed through a gel filtration column to determine if they were monomeric.

Figure 12 shows the gel filtratration profile of NBD1,,5-,,5 refoIded at pH 8.0

obtained with a Superose 12 column (Pharmacia). The profile indicates the

presence of a single monomeric species consistent with the size of NBD1.

Interestingly, al1 three peptides were eluted at the same volume (NBDl,,,,,

and NBDl,,,,, are not shown), indicating that they may have a similar

Stokes radius, despite having different molecular weights. Although the

peptides did not appear to aggregate (i.e. no elution at the exclusion volume),

it was noted with the NBDl,,,, and NBDl,,,-,, peptides that the relative

intensity of the elution peak appeared to decrease over time (days), suggesting

that the proteins may be aggregating/precipitating in large enough particles

that they were filtered through the column's pre-filter. However, the

intensity of the NBDl,,,-, peak remained constant even when the refolded

protein was incubated at room temperature for several days. This may

indicate that it was able to fold to a conformation that was a closer

approximation of the native domain than were the other two peptides.

Volume (ml)

Figure 12. Gel Filtration of NBD1,,5.,. NBDl,,,., (200~1, 1.5pIV.I) was applied to a Superose 12 (Pharmacia) gel filtration colwrin, equilibrated with Gel Filtration buffer (2OmM Tris, pH 8.0, 150 mM NaCI, 400 mM L-arginine). Absorbante was measured at 280 nrn to detect the elution of protein from the column. The elution volumes of molecular weight standards are indicated with arrows: blue dextran (2,000 kDa), BSA (66.4 kDa), and lysozyme (14.3 kDa). The column flow rate was 0.5 ml/min with a cliart speed of 2 mm/rnin.

O 0.5 1 1.5 2 2.5 3 3.5 4

[GdmClf (M)

Figure 13. Guanidinium Chloride Denaturation of NBDl Peptides. NBDl peptides were refolded to monomeric species at pH 8.0. NBDl,,,, (O); NBDI,,,,,, (X); and NBD1,,5.,,5, (+) were then diluted two fold with increasing concentrations of GdmC1, and incubated for 2 hours at room temperature (see Methods section 2.4). AG calculated for NBDl peptides were NBD14,,.5,,, 3.1 kcal/mol; NBDI,,,.,,, 4.3 kcal/mol; and NBDI ,,,.,, 5, 4.5 kcal/mol. Intrinsic tryp tophan fluorescence was measured by excitation at 280 nm and ernission at 335 nrn. The fraction of folded protein (ff) was calculated by the following equation: ff=(fluorescence-lower baseline)/(upper baseline-lower baseline) (see Methods section 2.4). (Raw data is shown in Appendix D)

Guanidine denaturation of NBDl peptides were characterized by

sigrnoidal curves, revealing CO-operative folding for each peptide. As shown

in Figure 13, the stability of 384-649 and 415-675 were comparable. However

the shortest peptide, 404-589, was notably less stable, as unfolding occurred at

considerably lower concentrations of denaturant. Such a reduction in stability

may indicate that the peptide is missing amino acids which are important for

the three dimensional structure of the dornain. The stability seen with the

- - -

amino and/or the carboxyl terminus. However, the stability seen with the

415-675 peptide supports that it is the carboxyl terminus which provided the

most benefit.

4.4. ATP Binding of NBDI,,,, NBD1384-6491 and NBDZ,,, Peptides

Azido-ATP binding experiments were performed by using 8-azido ATP

labelled with "P at either the a or the y phosphate. Azido analogues are

activated by UV light to produce a highly reactive nitrene group which forms

a covalent attachment to the binding site of the protein. The binding

experiments were performed on peptide solutions that had been refolded to a

concentration of 3 yM into refolding buffer (100 mM Tris, 400 mM L-arginine,

2 mM EDTA, pH 6, 7, or 8, as indicated). These protein solutions were diluted

two-fold in the azido-ATP binding experiments, resulting in a final peptide

concentration of 1.5 PM, and buffer conditions of 50 mM Tris, 200 mM L-

arginine, and 1 mM EDTA (pH 6,7, or 8, as indicated). Since the experiments

were typically completed within ten minutes after dilution, it was expected

that the protein would remain soluble and monomeric, even with the

reduced levels of L-arginine. It was later determined that some aggregation

occurred ai lower concentrations of L-arginine (to be discussed later in section

4.5.1).

4.4.1.1. NBDl peptides are labelled &y [ y32~] -8 -az ido -~TP

Azido-ATP binding experiments resulted in labelling of refolded NBDl

peptides. A typical labelling experiment for NBD1,,,4-5, is shown in Figure 14.

As depicted, this labelling could be cornpeted with various nucleotides and

did not occur when the protein was denatured with SDS or when the azido

reagent was quenched with DTT or pre-irradiated. In addition, there was no

non-specific labelling of BSA or carbonic anhydrase. It was however noted

that the extent of labelling was relatively weaker than what would be expected

for a protein with a binding constant in the micromolar range.

Another puzzling observation was that NBDl was labelled even in the

absence of UV irradiation. This 'dark' labelling was hypothesized to be

possibly due to the presence of a sulphydryl group in the vicinity of the ATP

binding site that was reducing the azido group (as occurs with the quenching

reagent, DTT). Consistent with this hypothesis was the characterisation of a

required cysteine residue in the first nucleotide binding domain of mouse P-

glycoprotein (Dayan et al., 1996). To explore this possibility, the two cysteine

residues common to al1 the NBDl peptides were replaced by site-directed

mutagenesis and these mutant peptides were assayed for their ability to bind

[?-"Pl-8-azido-ATP both in the presence and in the absence of UV irradiation.

The cysteine residues were mutated to both alanine and serine, as such

mutations were considered to be relatively conservative and would likely not

affect the folding of the protein, and both mutants at each site could be

L A - - - * - the cysteine-524 mutants (C524A and C524S) had no effect on the dark

labelling of NBD1. Similar results were also seen with cysteine-491 mutants,

C491A and C491S (not shown).

Figure 14. [y-32Pl-8-azido-ATP binding to NBDl,,,.,,,. The top and bottom panels represent the Coomassie stained gel and the autoradiograph, respectively. Al1 reactions were perfornied with NBD1,,,.58, (refolded at pH 8.0, at a final concentration of 1.5w) and [Y-~~P]-$- Azido-ATP (10pM). Al1 reactions, except that in lane 9, were irradiated for 10 minutes, in open eppendorf tubes, approx. 5 cm from a UV light box. Lane 1 represents NBDl and azido-ATP alone. Lanes 2, 3, 4, 12, and 13 represent NBDl that was pre-incubated for 5 min with 10 mM ATP, ADP, AMP, GTP and ATP-y-S, respectively. Lane 5 represents NBDl tliat was pre- treated with 2% SDS to denature the protein. In order to establish that the labelling of protein was specific, BSA and carbonic anhydrase were used as negative controls (lanes 6 and Il). Lane 7 represents the reaction with BSA and NBDl together. Lane 8 represents the reaction with azido-ATP tlnt liad been pre-irradiated, prior to addhg NBDl. Lane 9 represents NBDl and azido-ATP tliat was not irradiated. In lane 10, 20 mM DïT was added to quench the azido group so tliat it would not bind to NBD1.

the 'dark' labelling of NBD1, it was subsequently hypothesized that the

labelling of NBDl in the absence of UV irradiation might be the result of

phosphate tram fer due to contaminating phosphatases, or less likely, due to

the ATPase property of the NBDl peptide. This side reaction could possibly be

eliminated by using the [a-32P] labelled analogue of &azido-ATP.

Figure 15. [y-32P]-8-azido-ATP binding to NBDI,,,.,, C542A and C524S. The cysteine mutation has no effect an the dark reaction. NBDl,.,,-C524A and C524S mutants were refolded at pH 8, then assayed for their ability to bind [?/-32P]-8-azido-ATP, with and without W irradiation, or in the presence of 10 rnM DTï. NBDl,,,,, (1.5pM) was incubated with [Y- 32P] -8 -az ido -~~~ (5w) at 4°C in amber eppendorf tubes. The reactions were quenched with 5 X SDS-PAGE loading dye with 100 mM DïT and analysed on a 12% acrylamide gel. The gel was stained with Coomassie Brilliant Blue (not shown) and radioactivity was quantitaled by exposure on a phosphorimager screen.

Binding experiments were repeated using the [ w ~ ~ P ] analogue of 8-

azido-ATP (Figure 16) and the labelling of protein in the absence of UV

irradiation was significantly reduced. Furthermore, the use of [a-32P]-8-azido-

ATP also resulted in a much stronger labelling of NBDl than with the [ P r ]

labelled analogue (7 to 60 fold). This was determined by measuring the

intensity of the labelled band, taking into account the specific activity of each

analogue. This labelling was also dependant on pH, such that pH 6 > pH 7 >

pH 8 (approx. 10:3:1, respectively). Conversely the labelling with the [Y-~~P]

labelled analogue was independent of pH.

Although, the binding at pH 6 seemed strong, it was a puzzling that

this could only be seen with the [cx-~~P] analogue. Furthermore, significant

non-specific labelling was observed when a His-tagged c-jun leucine zipper

domain was used as a negative control. Such labelling also displayed the

same pH dependence as was seen with NBD1. These results raised the

concern that the labelling may not be specific and was instead some artefact

that was enhanced at lower pH.

Figure 16. Characterisation of the labelling of NBDl,,.,, by ld2P]-8-azido-ATP. 5pM [ a - 3 2 ~ ] - 8 - a z i d o - ~ ~ ~ or [ y - 3 2 ~ ] - & a z i d o - ~ ~ ~ was incubated with a His-tag fusion to the leucine zipper domain of c-jun (c-jun-LZ), or 1.5w NBDl,,.,, (refolded at pH 6.0, 7.0, or 8.0). Labelling with azido-ATP was competed with 10 mM ATP . Reactions were irradiated for 5 min on Coors white porcelain spot plates. Dark controls (no UV) were not irradiated. The reactions were quenched with 5X SDS-PAGE loading dye cmd analysed on a 12% acrylamide gel, by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue (not shown) and radioactivity was quantitated by exposure on a phosphorimager screen.

4.4.1.3. ATP does not compete for 8-azido-ATP binding.

In order to determine the specificity of the labelling by [a-3zP]-8-azido-

ATP, binding was performed in the presence of increasing concentrations of

ATP. It was found that extremely high levels of ATP were needed in order to

achieve inhibition of [a-32P]-8-azido-ATF labelling of NBDl (Figure 17). This

finding, in addition to the observation that c-jun had signifiant non-specific

labelling (Figure 16), led to the suspicion that the cornpetition may be non-

specific. In order to assess this possibility, c-jun-LZ and BSA were labelled

with [a-32P]-8-azido-ATP and cornpeted with 10 mM ATP. As shown in

Figure 18, 10 mM ATP caused a similar decrease in labelling of these two

proteins, as was seen with NBD1, indicating that the labelling was not specific

for ATP binding.

Figure 17. Cornpetition of [a-32P]-8-azido-ATP labelling of NBDl with ATP. The labelling of NBDl by [a-32P]-8-azido-ATP was competed with 5 ph4 to 20 mM ATP. Reactions were pre-incubated with the indicated concentration of ATP prior to incubation with [CX-~~P] -~ - azido-ATP. Reactions were irradiated for 5 min cm Coors white porcelain spot plates. The reactions were quenched with 5X SDS-PAGE loading dye and analysed un a 12% acrylamide gel, by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue (not shown) and radioactivity was quantitated by exposure on a phosphorimager screen.

Figure 18. Non-specific Labelling of Proteins by [~x-~~P]-8-azido-ATP. BSA and c-jm-LZ were incubated with 5pM [a-32P]-8-azido-ATP, in the presence or absence of 10 mM ATP. Reactions were irradiated for 5 min an Coors white porcelain spot plates. The reactions were quenched with 5X SDS-PAGE loading dye and andysed on a 12% acrylamide gel, by SDS- PAGE. The gel was stained with Coomassie Brilliant Blue (not shown) and radioactivity was quantitated by exposure on a phosphorimager screen.

As seen in Figure 18, there is increased of labelling of proteins at pH 6

cornpared to pH 8. It was also noted that upon incubation at 4'C, NBDl

peptides solutions began to precipitate at pH 6.0, became somewhat cloudy at

pH 7.0, but remained clear at pH 8.0. As shown in Figure 19, a decrease in the

concentration of L-arginine, a condition which was later shown to cause

NBDl,,,,, to aggregate (to be discussed in section 4.5), also resulted i n

. stronger labelling of protein. Of particular note was the strong signal seen at

the interface of the stacking and separating gels with c-jun-LZ. When this

experiment was performed, the c-jun-LZ preparation was three weeks old,

and had been stored at 4'C. Despite there being relatively little protein at the

interface, as determined by Coomassie staining (not shown), a strong signal

was detected. When the same solution was first clarified by centrifugation at

12000g for 20 min no labelling was observed for comparable levels of protein

(assessed by Coomassie staining, not shown).

aggregated - c-jun-LZ

4- c-jun-LZ

Figure 19. [c~-~V]-8-azido-ATP Labelling of Protein Aggregates. NBD13,-,, was refolded at pH 6.0 and diluted to 1.5 in pH 6.0 buffer containing 50 mM (lanes 1 and 2 ) or 400 mM (lanes 3 and 4) L-arginine. 20 mM ATP was added to one of each sample (lanes 2 and 4). 1.5 p M C-jun-LZ (three weeks old, 4"C), untreated (lane 5) or clarified by centrifugation (12000g, kane 6) was diIuted into pH 6.0 buffer containing 200 mM L-arginine. Samples were incubated with 5pM [~t-~~P]-8-azido-~TP and irradiated for 5 min on Coors white porcelain spot plates. The reactions were quenched with 5X SDS-PAGE loading dye and analysed on a 12% acrylarnide gel, by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue (not shown) and radioactivity was quantitated by exposure on a phosphorimager screen.

To establish that [a-32~]-8-azido-~~P was, in fact, preferentially labelling

aggregated peptide, NBDl,,,,, protein samples were centrifuged at 12000g

after irradiation, then analysed by SDS-PAGE (Figure 20). It was observed that

NBDI,,,,, showed increased precipitation at lower pH (and was almost

completely aggregated at pH 6.0).

supernatant

pellet

Figure 20. Autoradiograph of Ca-3ZP]-8-azido-ATP binding to NBDl,,,, .Refolded protein (1.5pM) was innibated with 5p.M [~~-9?]-8-azido-ATP ai pH 6.0, pH 7.0, and pH 8.0 (total vol. 40 pl), in the presence and absence of 5 rnM ATP. Reactions were incubated for 5 min on Coors white porcelain spot plates (on ice), then irradiated or left in the dark (no UV) for 5 min. The reaction mixtures were then transferred to 1.5 ml centrifuge tubes, and centrifuged a t 12,000g for 10 min. Half the supernatant (20~1) was added to 5p1 SDS-PAGE loading buffer, and electrophoresed on a 12% acrylarnide gel (top gels). The remaining supernatant plus the pellet was added to 5 pl SDS-PAGE loading buffer and electrophoresed on a separate gel. The gels were stained with Coomassie Brilliant Blue (R-250) and exposed on a phosphorimager screen.

4.4.2. Intrinsic Tryp tophan Fluorescence

Because of the problems associated with the 8-azido-ATP binding

experiments, it was necessary to find other assays for ATP binding. One of the

assays used looks for changes in intrinsic tryptophan fluorescence of the

L I I --

-- Q - - --a --- - -- -- --- J ------- -

conformational change in the three dimensional structure of the protein,

thereby changing the chemical environment of buried tryptophan residues,

and thus changing their fluorescence characteristics. Another advantage in

using this assay is that it removes the need to use an analogue of the ligand

that possesses sorne measurable characteristic. Instead, the measurable

characteristic lies within the protein itself, and binding experiments can be

used with the true ligand (in this case, ATP).

ATP was added at concentrations from 10-400 PM, and the intrinsic

tryptophan fluorescence of al1 three NBDl peptides was measured. However,

there were no detectable changes in tryptophan fluorescence. Although, this

assay did not detect ATP binding, it was not sufficient to suggest that the

protein was not capable of binding ATP, as the conformational change may

have been subtle. Therefore, another assay was used.

4.4.3. TNP-ATP

TNP-ATP is an ATP analogue that displays an enhancement in

fluorescence when the ligand is buried with the binding site of the protein.

However, due to the hydrophobic nature of the trinitrophenyl group, it

typically binds to ATP binding proteins with much higher affinity (as much as

1000 fold). It is consequently not an ideal ligand, but is attractive because of

the ease in performing the binding experiments.

O 5 10 15

[TN P-ATP] (PM)

Figure 21. TNP-ATP Binding to NBDl,,.,,,. Binding assay was performed as described (Methods section 3.2). The symbols are as follows: TE buffer (O), BSA in TE (i), refolding buffer ( ), BSA in pH 8.0 refolding buffer ( x ) , NBDl,,,.,, in 4M GdmCl (O),NBDl,,,. 5,, in pH 8.0 refolding buffer (+). (Raw data is shown in Appendix E)

TNP-ATP binding assays were performed on NBDl,,,,, and NBDl,,,

,,, peptides that had been refolded by rapid dilution in the presence of 400

mM L-arginine. As seen in Figure 21, no significant enhancement of

fluorescence was detected with NBDl,,,,,, in refolded buffer with arginine

above that observed with NBDl,,,,,, in 4 M GdmC1. Similar results were

seen with NBDl,,,,,. It was also noted that BSA exhibits a significant

enhancement of fluorescence in T:E buffer, whereas the fluorescence

enhancement with BSA was greatly reduced in arginine buffer. Despite the

significant fluorescence enhancement seen with BSA, this enhancement

could not be competed with ATP, indicating that the binding was not specific

1 A. A

presence of 0.4 M L-arginine effectively abolished the fluorescence

enhancement, and could conceivably be having the sarne effect on NBD1.

The effect of L-arginine on TNP-ATP binding was further explored by

studying adenylate kinase (Sigma). Adenylate kinase is a protein with a well

characterized nucleotide binding fold that has been used as a mode1 for NBDl

(Hyde et al., 1990), and was therefore used as a positive control for ATP

binding. Adenylate kinase was capable of binding to TNP-ATP in the

micromolar range and this binding could be competed with ATP (not shown).

As shown in Figure 22, the binding of TNP-ATP to adenylate kinase

was markedly reduced as the concentration of L-arginine was increased. This

effect was not noticed with high concentrations of salt, and was probably the

result of a change in conformation of the protein to such an extent as to

prevent function. This interference was presumably also occurring with the

NBDl peptides, which needed high concentrations of L-arginine to remain

monomeric. It was therefore decided to explore the effect of different

concentrations of L-arginine on the aggregation of NBDl peptides.

O 2 4 6 8 10

[TNP-ATP] (FM)

Figure 22. Effect of L-Arginine on TNP-ATP Binding to Adenylate Kinase. Adenylate kinase (1.8 PM) was incubated with the indicated concentrations of TNP-ATP in the presence of O mM (O), 40 rnM (A), 100 mM (+), 200 mM (X), or 400 mM (+) L-Arginine. Fluorescence enhancement was measured as described (see Methods section 3.2) by excitation at 408 nrn and emission at 538 nm. (Raw data is in Appendix F)

4.5. Aggregation of NBDl Peptides.

The aggregation of NBDl peptides was studied by measuring light

scattering of peptide solutions upon dilution from 6 M GdmCl into different

refolding buffers. It is based on the principle that as aggregated protein

particles increase in size, more light is reflected in al1 directions.

4.5.1. High Concentrations of L-Arginine Prevent Aggregation of NBDl

Peptides.

Al1 three NBDl peptides remained soluble upon rapid dilution (3 vM)

into buffer containing high concentratioiis (400 mM) of L-arginine. As shown

of L-arginine resulted in increased light scattering. After incubation

overnight at 4'C, some cloudiness was observed at pH 7.0 and precipitation

was seen pH 6.0, indicating that the peptides were less soluble at lower pH.

O 1 00 200 300 400 500 time (sec)

Figure 23. Light Scattering of NBD1,,,.,,,. NBD1,0,.B9 (in 6 M GdmCl) was rapidly diluted to 3 pM final protein concentration (1 O0 mM Tris, pH 8.0; 2n1M EDTA) in the presence of 400 mM (O), 200 mM ( ), 100 mM (O), or O mM (X) L-arginine at 4°C. Light scattering (400 run) was measured as described in Methods, section 2.5. (Raw data is in Appendix B)

O 1 O0 200 300 400 500 time (sec)

Figure 24. Light Scattering of NBDl,,,.,,. NBDl,,,,, (hi 6 M GdmC1) was rapidly diluted to 3 pM final protein concentration (100 mM Tris, pH 8.0; 2mM EDTA) in the preserice of 400 niM (O), 200 mM ( ), 100 nSVI (O), or O niM (X) L-arginine at 4°C. Ligl~t scattering (400 nm) was measured as described in Methods, section 2.5. (Raw data is in Appendix B)

O 1 O0 200 300 400 500 time (sec)

Figure 25. Light Scattering of NBDl,,,.,,,. NBD1J15.1r75 (in 6 M GdmCl) was rapidly diluted to 3 LLM final y rotein concentration (100 mM Tris, pH 8.0; 2mM EDTA) in the yreseiice of 400 mM (O), 200 111M ( ), 100 mM (O), or O niM (X) L-arginine at 4°C. Light scattering (400 nm) was n-icrtsured as described in Methods, section 2.5. (Raw data is in Appeiidix 6)

The effect of pH on protein solubility was further explored by refolding

the three NBDl peptides at pH 6, 7, and 8. There was a pronounced effect on

the aggregation of NBDl,,,, when the peptide was refolded at lower pH. As

shown in Figure 26, the peptide aggregated at pH 7, and within seconds at pH

6. Similar, although less pronounced effects were seen with the other two

peptides (see Appendix C).

O 500 1 O00 1500 2000 tirne (sec)

Figure 26. Effect of pH on the Light Scattering of NBDI,,,.,,. NBDl,,.,,, (in 6 M GdmC1) was rapidly diluted to 3 pM final protein concentration at pH 6.0 (O), pH 7.0 ( ), and pH 8.0 (0) at 30°C. Light scattering (400 nm) was measured as described in Methods section 2.5. (100 rnM Tris, pH 8.0; 2mM EDTA) (Raw data is in Appendix A)

Conclusions and Future Directions

The initial intent of this study was to isolate a single domain from a

multidomain protein, establish a functional assay for this domain, and to

study the effects of mutations on the domain's function. That this could be

achieved was based on the occurrence of independent proteins that provide

the MSD and NBD components of bacterial ABC transporters, and the

modular design of ABC transporters in higher organisms. The attempts to

obtain purified soluble protein from E. coli even with a variety of refolding

strategies, revealed that NBDl of CFTR is extremely insoluble.

1. L-Arginine Prevents Protein Aggregation

In this study, and in studies by another group (Qu and Thomas, 1996;

Qu et al., 1997), it was found that NBDl peptides could only remain soluble

and monomeric in the presence of high concentrations (400 mM) of L-

arginine. L-arginine acts as a mild denaturant capable of shielding non-

specific interactions. It was hoped that the arginine would only be needed

during the refolding process, and could be removed after the protein had

achieved a folded siate. There are examples in which high concentrations of

L-arginine (0.4-0.8 M) have been used successfully to allow correct protein

folding, but could later be removed by dialysis to leave functional protein

(Garboczi et al., 1992; Stoyan et al., 1993). It is evident that this is not possible

with the NBDl peptides examined.

Perliaps the primary reason for the observed insolubility of NBDl is

that the actual domain boundaries have yet to be firrnly established. If an

essential portion of NBDl is missing, or if an unstable portion of an adjacent

domain is present, then this could lead to the exposure of hydrophobic

surfaces and u 1 tirna tely, protein aggrega tion.

Furtlier it is conceivable that even under conditions in which the

correct domain boundaries are employed, NBDl could still be relatively

insoluble. CFTR is a membrane protein, with multiple domain-domain

interactions, and thus the isolation of a single domain from the rernainder of

the protein and from the membrane may deprive the peptide of required

hydrophobic associations. In the absence of these associations, the peptide

would compensate wi th non-specific aggregation. These associations would

have to be mimicked by fusions or by solvent modifications to obtain a

soluble domain.

Models for NBDl were proposed, based on sequence alignments to

other nucleotide binding folds soon after the cloning of the gene (Hyde et al.,

1990) and more recently by other groups (Annereau et al, 1997; Hoedemaker et

al., 1997). Althougli the models share some similarities in some of their

structural aspects, they are based on different proteins and there is not

complete agreemeni on the domain boundaries or the absolute alignment.

However, they do provide testable start points to acliieve an accurate rnodel,

and i t may be possible to predict which hydrophobic amino acids needed

by rational site directed mutagenesis and tested to verify that they lead to a

more soluble peptide whose function could then be studied.

3. NBDl extends into the putative R domain

The two most recent rnodels of NBDl of ABC transporters include

extension at the carboxyl terminus over previous models. In CFTR, this

includes the most highly conserved segment of the R domain. Althougli the

actual domain boundaries of NBDl are not known, it is Iikely that the C-

terminal boundary does extend into the putative R domain. This has been

supported by three observations. First, the stability of the NBDl,,,-,, and

NBDl,,,,, peptides, as determined by guanidinium chloride denaturation,

are significantly higher than that of the NBDl,,,,, peptide. Secondly,

NBDl,,,,,, was observed to remain soluble for extended periods of time and

at elevated temperatures, as compared to the other two peptides. And finally,

poorer expression and degradation were noted wlien expressing NBDl,,,,,,

and pETNBD1 /R348-858 peptides. These effects were most strongly iioted with

the pETNBDl/R,,,,, peptide which extends to include the entire R domain

and was only detectable by Western blot. This could be an indication that the

peptide was less prone to aggregation (and subsequently, inclusion body

formation) and tl-ierefore more susceptible to the degradation processes of the

bacterial liost.

boundaries better than any other peptide known to date, it still remains

susceptible to aggregation under physiological conditions, and therefore is of

limited use in studying ATP binding. New approaches for finding soluble .. .

peptides are needed. It may be possible to devise an 'assay' for solubility of

random protein fusion sets where individual fusions can be functionally

selected. For example, this may be achieved witl-t fusions to an antibiotic

resistance gene and subsequent screening for difference in resistance levels

(A. Davidson, J. Forman-Kay; persona1 communication).

4. ATP Binding

Although NBDl is certainly involved in ATP binding/hydrolysis i n

the full lengtl-i protein, we have not been able to detect ATP binding in the

isolated peptide. The ATP analogue, 8-azido-ATP, displayed some binding,

but this was later found to be an artefact of peptide aggregation. To achieve

nucleotide binding, the NBDl peptides would need to be folded in a state at

least somewhat comparable to its native conformation in the multidomain

CFTR. While it was clearly possible to acl-tieve a folded state of monomeric

peptide in L-arginine tl-tat possessed an ordered structure, binding did not

occur. These results do not suggest that NBDl does not bind ATP, but rather

that the conditions required in order to keep the protein soluble and

monomeric interfere with the ability to study ATP binding. This is supported

by the specific binding of adenylate kinase to TNP-ATP which was observed to

(Figure 22).

It should be noted that Qu et al. (1997) have reported relatively strong

ATP binding of their peptide (amino acids 404-589), by intrinsic tryptophan

fluorescei~ce, even in the presence of 0.4 M L-arginine. Such strong binding

was surprising, because we observed that 0.4 M L-arginine had an

overwhelming negative effect on the binding of TNP-ATP to adenylate

kinase, seen with Our positive control protein. The Kd is also interesting as it

is much stronger than was observed in studies of the full length protein

(Travis et al., 1993).

5. Protein folding

A more interesting aspect of the study by Qu and Thomas indicated

that NBD1-AF508 has a higher rate of aggregation at higher temperatures, and

was used to argue that this may directly reflect reduced folding efficiency i n

vivo. This was supported by showing that the aggregation and misfolding

could be reduced in the presence of glycerol, and that the AF508 revertant

mutation, R553M, could correct the AF508 folding defect (Qu et al., 1997). It

had previously been demonstrated that many NBDl mutants, result in a

failure to produce mature CFTR (Gregory, et al., 1991). At least some of these,

including CFTR-AF508, were shown to mature with addition of reagents or

conditions which facilitate correct folding. By studying the tendency of O ther

NBDl mutant peptides to aggregate, it may be found that their mechanism of

A A . L I I H I C ~ C I " L ' *Y H I Y V 4--A-+- -Y -CI---- - - - r"r"'J "A--- " - - - W . "'W.,., "b"""1'

may lead to further indications to focus on protein folding as a possible

therapy for the majority of cases of cystic fibrosis.

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Appendices

1. Appendix A. Raw Data for Light Scattering of NBDl Peptides at different PH- time (sec)

O 10 20 30 40 50 60 70 80 90 1 O0 110 120 130 140 150 1 GO 170 1 80 190 200 21 0 220 230 240 250 260 270 280 290 300 3 1 O 320 330 340 350 360 370 360 390 400 4 10 420 430 440 450 4 60 4 70 4 S0 490 500 510 5 20 530 510 5,717 5 GO 5 70

2. Appendix B. Raw Data of Light Scattering of NBDl Peptides Refolded in Varying Concentrations of L-Arginine.

time (sec)

O 2 4 5 6 8 10 12 14 15 16 18 20 22 24 25 26 2s 30 32 33 35 36 3 s 40 -1 1 44 45 4 6 48 50 52 54 55 56 58 60 62 64 65 66 6s 70 72 74

(sec) 404- 384- 415- 589 649 675

(sec) 404- 384- 415- 589 649 675

528 530 532 534 535 536 538 5-10 542 514 545 9 6 518 550 552 5 3 535 536 558 560 562 564 565 566 56s 5x1 577 574 575 576 578 3 0 553 3-1 5S5 5S6 555 39U 592 ,594 393 596 59s 600 602 604 605 606 60s 610 612 614 61.4 hlh 61s 620 612 624 6 3 626 62s 6.30 632 il 34 (33.5

636 6.3

lllll~ 1 K J fll&

(sec) 404- 384- 415- 589 649 675

LUI,

404- 589

-.-m.- . .., -5 (sec) 404- 384- 415-

589 649 675

O 500 1 O00 1500 2000 time (sec)

O 500 1 O00 1500 2000 time (sec)

Effect of pH on the tight Scattering of NBDl Peptides. NBDl,,.,,, (top panel) and NBDI,,,.,,, (bottom panel) were rapidly diluted to 3 final protein concentration at pH 6.0 (O), pH 7.0 ( ), and pH 8.0 (0) at 30°C. Liglit scattering (400 nm) was measured as described in Metl-iods section 2.5. (100 mM Tris, pH 8.0; 2mM EDTA) (Raw data is in Appendix A)

4. Appendix D. Raw Data of the Guanidium Chloride Denaturation of NBDl Peptides.

[GdmCI] (M) fluorescence 384-649 41 5-675

101.5 168

5. Appendix E. Raw Data of TNP-ATP Binding to NB 1NM-,8,e

[TN P-ATP] (PM)

TE buffer

BSA in TE refolding BSA in pH 8.0 NBDl,,,,,, in NBDl,,,,,, in pH 8.0 buffer refolding buffer 4M GdmCl refolding buffer

6. Appendix F. Raw Data of TNP-ATP Binding to AdenyIate Kinase

[TNP-ATP] buffer ADK buffer ADK buffer ADK (PM) no Arg no Arg 40 mM Arg 40 mM Arg 100 mM Arg 100 rnM Arg

[TNP-ATP] buffer ADK buffer ADK (FM) 200 mM Arg 200 mM Arg 400 rnM Arg 400 mM Arg

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