Bacterial Periplasmic Binding Proteins as Biosensors in Liposomes

Lab Meeting:

“Turning Houseflies into Fireflies”

Szostak Lab Howard Hughes Medical Institute, and Department of Molecular Biology, Massachusetts General Hospital,

Boston, Massachusetts 02114

Problem:Problem:• How can one track the locations and How can one track the locations and

relative concentrations of molecules relative concentrations of molecules (esp. those of prebiotic interest) in (esp. those of prebiotic interest) in liposomes?liposomes?

http://bioweb.wku.edu/courses/biol22000/2Bonds/http://bioweb.wku.edu/courses/biol22000/2Bonds/http://bioweb.wku.edu/pix/Pix.htmhttp://bioweb.wku.edu/pix/Pix.htm

Background:Background: Biosensors Biosensors

• Indicate the presence of moleculesIndicate the presence of molecules

• Specificity to targets & signal transduction:Specificity to targets & signal transduction: ligand ligand binding binding detectable physical changes detectable physical changes

• Should be reagentless:Should be reagentless: do not change composition as do not change composition as a consequence of making the measurementa consequence of making the measurement

• Ligand-Specific Periplasmic Binding ProteinsLigand-Specific Periplasmic Binding Proteins

– Superfamily of macromolecules that facilitate the active Superfamily of macromolecules that facilitate the active transport of ions, sugars and amino acidstransport of ions, sugars and amino acids

– Can be engineered to sense these substratesCan be engineered to sense these substrates

Background:Background: E. coliE. coli bPBPs bPBPs• bbacterial acterial PPeriplasmic eriplasmic BBinding inding

PProteinsroteins

– 2 domains linked by a hinge region 2 domains linked by a hinge region

– Ligand-binding site is located at the Ligand-binding site is located at the interface between them.interface between them.

• 2 conformations that interconvert via 2 conformations that interconvert via hinge bendinghinge bending

– Ligand-free openLigand-free open

– Liganded closedLiganded closed

• Conformational change attributed to Conformational change attributed to reporter functionsreporter functions

• Fluorophore Fluorophore (Acrylodan)(Acrylodan) conjugates conjugates of bPBPs specific for various ligandsof bPBPs specific for various ligands

Felder, C.B., Felder, C.B., et al.et al. (1999) (1999) AAPS PharmSci.AAPS PharmSci. 1 (2). 1-20. 1 (2). 1-20.

www.botgard.ucla.edu/. ../b0567tx.html

http://www.botgard.ucla.edu/html/botanytextbooks/lifeforms/insectivorousplants/b0567tx.html

http://www.botgard.ucla.edu/html/botanytextbooks/lifeforms/insectivorousplants/b0567tx.html

Background:Background: E. coliE. coli bPBPs bPBPs• Changes in fluorescence intensity Changes in fluorescence intensity

indicate ligand bindingindicate ligand binding

– Environmentally-sensitive fluorophores Environmentally-sensitive fluorophores are placed in locations that undergo are placed in locations that undergo conformational change (local conformational change (local global) global)

– Cys mutations are placed at these sites Cys mutations are placed at these sites so that changes in fluorescence may be so that changes in fluorescence may be directly linked to ligand binding.directly linked to ligand binding.

• Sites identified by De Lorimier Sites identified by De Lorimier et al.et al.

– Protein Science Protein Science (2002), 11:2655–(2002), 11:2655–2675.2675.

– Closed structure examination, open Closed structure examination, open vs. closed & structural/sequence vs. closed & structural/sequence homologyhomology

HingeHinge Mutated ResidueMutated Residue Bound LigandBound Ligand

De Lorimier De Lorimier et al.et al. Protein Science.Protein Science. 11:2655–2675. 11:2655–2675.

ABC Transport System:ABC Transport System:• AATP TP BBinding inding CCassetteassette

– Periplasmic Protein-Dependent Transport System (G-)Periplasmic Protein-Dependent Transport System (G-)– Binding Lipoprotein-Dependent Transport System (G+)Binding Lipoprotein-Dependent Transport System (G+)

• bPBPs are initial receptors in active transport across cellular bPBPs are initial receptors in active transport across cellular membranes &/or chemotaxismembranes &/or chemotaxis

• Each binds a specific substrate (e.g. sugar, amino acid, or ion) Each binds a specific substrate (e.g. sugar, amino acid, or ion) with high affinity. with high affinity.

• 3 Types of Constituents:3 Types of Constituents: Genes typically found in an operonGenes typically found in an operon– 2 integral membrane proteins (permeases) each having 6- 2 integral membrane proteins (permeases) each having 6-

transmembrane segmenttransmembrane segment– 2 peripheral membrane proteins that bind & hydrolyze ATP (most 2 peripheral membrane proteins that bind & hydrolyze ATP (most

evolutionarily conserved)evolutionarily conserved)– A periplasmic (or lipoprotein) substrate-binding protein A periplasmic (or lipoprotein) substrate-binding protein (the part we seek (the part we seek

to exploit in our assays) to exploit in our assays)

Labeling with AcrylodanLabeling with Acrylodan• 6-acryloyl-2-6-acryloyl-2-

dimethylaminonaphthalene dimethylaminonaphthalene

• Molecular Formula:Molecular Formula: C C1515HH1515NO NO

• Molecular Weight:Molecular Weight: 225.29 225.29

• Labeling Rxn:Labeling Rxn:

– 36.4 μM protein stock (0.014 g/ml) 36.4 μM protein stock (0.014 g/ml) for 300 μL proteinfor 300 μL protein

– Add 2.16 μl of 50 mM TCEP (50 Add 2.16 μl of 50 mM TCEP (50 mM TCEP; 10 mM dye)mM TCEP; 10 mM dye)

– Add 10.8 μl of 10 mM dye Add 10.8 μl of 10 mM dye (0.001g/0.5ml)(0.001g/0.5ml)

– Leave mixture in fridge overnight.Leave mixture in fridge overnight.

Emission:Emission: 500 nm 500 nmExcitation:Excitation: 390 nm 390 nm

Background:Background: Biosensors Biosensors

• Objective:Objective: Synthesize a Synthesize a collection of binding collection of binding proteins that exploits a proteins that exploits a common signal common signal transduction mechanismtransduction mechanism

– Fluorophore reports Fluorophore reports binding of specific binding of specific molecular targets in molecular targets in liposomesliposomes

– Amino acids, cations, Amino acids, cations, anions, dipeptides & anions, dipeptides & sugarssugars

Background: Biosensors

• Objective: Synthesize a collection of binding proteins that exploits a common signal transduction mechanism

– Fluorophore reports binding of specific molecular targets in liposomes

– Amino acids, cations, anions, dipeptides & sugars

http://www.darnellworks.com/a52/nr0007.htm

Binding Proteins of InterestProtein Size Binding ProteinBinding Protein GeneGene MutationsMutations

33.19633.196 kD ArabinoseArabinose araFaraF K301C, C64AK301C, C64A

57.536 kD57.536 kD DipeptideDipeptide dppAdppA D450CD450C

29.714 kD 29.714 kD IronIron fhuDfhuD

24.988 kD24.988 kD GlutamineGlutamine glnHglnH Y163CY163C

31.000 kD31.000 kD Glutamate-AspartateGlutamate-Aspartate gltI/ybeJgltI/ybeJ F126CF126C

17.822 kD17.822 kD Glucose-GalactoseGlucose-Galactose mglBmglB H152CH152C

26.141 kD26.141 kD HistidineHistidine hisJhisJ V163CV163C

40.775 kD40.775 kD MaltoseMaltose malEmalE D95CD95C

34.473 kD34.473 kD PhosphatePhosphate phoSphoS S164C (S197C)S164C (S197C)

28.460 kD28.460 kD RiboseRibose rbprbp A234CA234C

34.470 kD34.470 kD SulfateSulfate sbpsbp L65CL65C

Binding Proteins of InterestStatusStatus Protein Size Binding ProteinBinding Protein GeneGene MutationsMutations

11stst of double of double mutantmutant

33.19633.196 kD ArabinoseArabinose araFaraF K301C, C64AK301C, C64A

PurifiedPurified66.8982 kD66.8982 kD DipeptideDipeptide dppAdppA D450CD450C

Cloned WTCloned WT 29.714 kD 29.714 kD IronIron fhuDfhuD E203CE203C

Cloned WTCloned WT24.988 kD24.988 kD GlutamineGlutamine glnHglnH Y163CY163C

Expressed 31.000 kD31.000 kD Glutamate-AspartateGlutamate-Aspartate gltI/ybeJgltI/ybeJ F126CF126C

Functional33.334 kD33.334 kD Glucose-GalactoseGlucose-Galactose mglBmglB H152CH152C

FunctionalFunctional 59.239 kD59.239 kD HistidineHistidine hisJhisJ V163CV163C

Functional40.928 kD40.928 kD MaltoseMaltose malEmalE D95CD95C

FunctionalFunctional35.832 kD35.832 kD PhosphatePhosphate phoSphoS S164C (A197C)S164C (A197C)

Functional 28.474 kD28.474 kD RiboseRibose rbprbp A234CA234C

FunctionalFunctional 37.696 kD37.696 kD SulfateSulfate sbpsbp L65CL65C

phoSphoS:: Cloning with pET100/TOPO Cloning with pET100/TOPO• ChampionChampionTMTM pET Directional pET Directional

TOPO® Expression Kit TOPO® Expression Kit (Invitrogen)(Invitrogen)

Digest Confirmation GelDigest Confirmation GelSequenced & ExpressedSequenced & Expressed

↓↓

PhoS:PhoS: S164C (A197C) S164C (A197C)

PhoS:PhoS: Mass Mass SpectrophotometrySpectrophotometry• PhoS = 35.832 kDPhoS = 35.832 kD• His-Tag = 3 kDHis-Tag = 3 kD

38,832 D 38,832 D

hisJhisJ:: Cloning with pET100/TOPO Cloning with pET100/TOPO

HisJHisJ: V163C: V163C

↓↓


HisJ Purification ProcessHisJ Purification Process• 1L culture1L culture

• Ni-NTA columnNi-NTA column

• Dialysis:Dialysis: 50 mM HEPES, 50 mM HEPES, 50 mM NaCl, pH = 7.450 mM NaCl, pH = 7.4

• M.W. = 26 kDM.W. = 26 kD

• Labeled with AcrylodanLabeled with Acrylodan

50 kD50 kD

Affinity of HisJ:Affinity of HisJ: Titrations Titrations

• 20 mM MOPS, 100 20 mM MOPS, 100 mM NaCl pH 6.9mM NaCl pH 6.9

• 20 mM NaH20 mM NaH22POPO44, 100 , 100

mM NaCl pH = 6.9mM NaCl pH = 6.90

5

10

15

20

25

30

35

40

0 20 40 60 80

[HisJ] (mM)

emis

sio

n

sbpsbp:: Cloning with TOPO/pET100 Cloning with TOPO/pET100

Sbp:Sbp: L65C L65CDigest Confirmation GelDigest Confirmation GelSequenced & ExpressedSequenced & Expressed

↓↓

Sbp Purification ProcessSbp Purification Process

• 1L culture1L culture

• Ni-NTA columnNi-NTA column

• Dialysis:Dialysis: 20 mM Tris- 20 mM Tris-HCl, pH = 8.0, HCl, pH = 8.0, cholestryramine cholestryramine (Dowex 1X2-100) resin(Dowex 1X2-100) resin

• M.W. = 35 kDM.W. = 35 kD

• Labeled with AcrylodanLabeled with Acrylodan

rbprbp:: Cloning with TOPO/pET100 Cloning with TOPO/pET100

Rbp:Rbp: A234C A234CDigest Confirmation GelDigest Confirmation GelSequenced & ExpressedSequenced & Expressed

↓↓

Rbp Purification ProcessRbp Purification Process• ExpressedExpressed• Ni-NTA Ni-NTA

columncolumn• M.W. = 28 kDM.W. = 28 kD• Found in Found in

pellet?pellet?– Run through Run through

column with column with ureaurea

– May need May need refolding…refolding…

And the Others? More of the same…And the Others? More of the same…

SbpSbpRbpRbp

HisJHisJ

MglBMglB

MalEMalE

What can be eliminated:What can be eliminated:

• Buffer pH is not near pIBuffer pH is not near pI• Storage (never frozen, tested immediately Storage (never frozen, tested immediately

& still dysfunctional; also tried 5 & 30% & still dysfunctional; also tried 5 & 30% glycerol when frozen)glycerol when frozen)

• Sequences (Mutation clearly sequenced & Sequences (Mutation clearly sequenced & no other errors)no other errors)

• Mass (Mass Spec/SDS-PAGE)Mass (Mass Spec/SDS-PAGE)• Column contamination (each protein has Column contamination (each protein has

it’s own Ni-NTA column)it’s own Ni-NTA column)

SDS-PAGESDS-PAGE

Still More Proteins…Still More Proteins…

DppA:DppA: Dipeptide Binding Dipeptide Binding Protein (D450C)Protein (D450C)


↓↓

DppA:DppA: D450C Mutation D450C Mutation• Very difficult to sequence Very difficult to sequence

mutagenized segment (N’s); 3 mutagenized segment (N’s); 3 different sequencing primers!different sequencing primers!

• Expressed to test functionality, Expressed to test functionality, SDS-PAGE & M.S.SDS-PAGE & M.S.


MalE:MalE: Maltose Binding Maltose Binding Protein (D95C)Protein (D95C)


↓↓ ↓↓


MglB:MglB: Glucose-Galactose Glucose-Galactose Binding Protein (H152C)Binding Protein (H152C)


↓↓ ↓↓


YbeJ (GltI):YbeJ (GltI): Glutamate- Glutamate-Aspartate Binding Protein Aspartate Binding Protein (H152C)(H152C)


↓↓


GlnH:GlnH: Glutamine Binding Glutamine Binding Protein (Y163C)Protein (Y163C)

FhuD:FhuD: Fe Fe3+3+ Binding Binding ProteinProtein

Future PlansFuture Plans• High Priority:High Priority:

– Sbp, Rbp, MglB, MalE & Sbp, Rbp, MglB, MalE & HisJ:HisJ: Repair defective Repair defective binding measurementbinding measurement

• Investigate effect of salts, pH Investigate effect of salts, pH (unlikely)(unlikely)

• Possible contamination?Possible contamination?

– DppA:DppA: Test kinetics (M.S.) Test kinetics (M.S.)

– PhoS:PhoS: Keep working or buy? Keep working or buy?

• Lower Priority:Lower Priority:

– AraF:AraF: C64A (K301C done) C64A (K301C done)

– YbeJ:YbeJ: F126C F126C

– GlnH:GlnH: Y163C Y163C

– FhuD:FhuD: E203C E203C

• Devise a new assay Devise a new assay system?system?

Concerns:Concerns:- Cost to produce - Cost to produce “in-house”“in-house”- StabilityStability- Ease of use/time Ease of use/time to produce to produce- System limitationsSystem limitations

AcknowledgmentsAcknowledgments

• JackJack

• Sheref Sheref

• MarkMark

• RaphaelRaphael

• YolletteYollette

• FlorianFlorian

??Questions????Questions??

Technology

Bacterial Periplasmic Binding Proteins as Biosensors in Liposomes