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From model membranes to bacterial pathogenesis: Investigations of
membrane protein structure and dynamics
Linda ColumbusUniversity of Virginia
Department of Chemistry and Department of Molecular Physiology and Biological Physics
Overview of the structure and function of membrane proteins
OM
IM
BtuB LamB OmpX
KcsA SecYEG BtuCD
≈ 150 polytopic membrane protein structures deposited in the pdb compared to >37,000 soluble protein structures
≈ 55 % of bacterial membrane proteins have unknown functions≈ 60% of drugs on the market are targeted to membrane proteins
Stabilizing membrane proteins for structural and functional
studies
Investigating the structure and function of bacteria membrane
protein – host interactions
Developing quantitative spectroscopic methods for
investigating membrane protein structure and function
Biophysical methodsNMREPR
SAXSCrystallography
Kroncke, Horanyi, and Columbus Biochemistry 49:10045 (2010)
Detergent micelles are typically used to extract and stabilize membrane proteins
6
Detergents used for NMR polytopic membrane protein structure determination
dodecylphosphocholine
OmpG PagP OmpA
DsbB
PNAS 99 (2002)PNAS 104 (2007)
DAGK
Science 324 (2009)
NSB. 8 (2001)
Mol. Cell 31 (2008)
octyl glucoside
PagP
PNAS 101 (2004)
Lauryl-dimethylamine-oxide
VDAC
Science 321 (2008)
OmpX
1, 2-di-cn-sn-glycero-phosphocholine
PNAS 98 (2002)
pSRII
NSMB. 17 (2010)
n=7 n=6
25 Å
Detergents influence the completeness of NMR observations and tertiary interactions
DM
DDM
FC-10
FC-12
1. What process in DDM and FC-10 is contributing to line broadening?
2. Why do DM and FC-12 PDCs have reduced line broadening?
Protein Science 15, 961 (2006)
Site-directed spin labeling
methanethiosulfonatespin label
R1
Disordered
Weakly Ordered
Strongly Ordered
Modulation of nitroxide dynamics by backbone fluctuations and tertiary interactions
20 G
Biochemistry 35, 7692 (1996)
SDSL indicates the helical interaction is disrupted in FC-10
JACS 131, 7320 (2009)
Detergents influence the completeness of NMR observations and tertiary interactions
DM
DDM
FC-10
FC-12
1. What process in DDM and FC-10 is contributing to line broadening?
2. Why do DM and FC-12 PDCs have reduced line broadening?
Protein Science 15, 961 (2006)
15N (ppm
)
DoDM 104
108
112
116
120
124
128
9 8 71H (ppm)
FC-10
9 8 7
104
108
112
116
120
124
128
Detergent (abbreviation) Ag # L(Å)
Shperoid rHC
(Å)VHC
(nm3)
Ionic property
n-decyl--D-maltoside (DM) 80-90 34 oblate 12, 23 26 Non-ionic
n-dodecyl--D-maltoside (DDM) 135-145 39 oblate 14, 29 49 Non-ionic
n-decylphosphocholine (FC-10) 45-53 28 prolate 21, 13 15 zwitterionic
n-dodecylphosphocholine (FC-12) 65-80 34 prolate 26, 16 27 zwitterionic
Detergent (abbreviation) Ag # L(Å)
Shperoid rHC
(Å)VHC
(nm3)
Ionic property
n-decyl--D-maltoside (DM) 80-90 34 oblate 12, 23 26 Non-ionic
n-dodecyl--D-maltoside(DDM) 135-145 39 oblate 14, 29 49 Non-ionic
n-decylphosphocholine (FC-10) 45-53 28 prolate 21, 13 15 zwitterionic
n-dodecylphosphocholine (FC-12)
65-80 34 prolate 26, 16 27 zwitterionic
DDM/FC-10(molar ratio 4:7)
77(30/47)
34 oblate 14, 21 26 zwitterionic
Geometry of micelle influences the completeness of NMR observations and tertiary interactions
JACS 131, 7320 (2009), J. Phys Chem. 111,12427 (2007)
oblate
prolate
What about another detergent mixture?
JACS 131, 7320 (2009)
Unique structure is stabilized by a hydrophobic match to the detergent shape and size
DM, FC-12, and FC-10/DoDM
DoDM
FC-10
15
Detergents used for NMR polytopic membrane protein structure determination
dodecylphosphocholine
OmpG PagP OmpA
DsbB
PNAS 99 (2002)PNAS 104 (2007)
DAGK
Science 324 (2009)
NSB. 8 (2001)
Mol. Cell 31 (2008)
octyl glucoside
PagP
PNAS 101 (2004)
Lauryl-dimethylamine-oxide
VDAC
Science 321 (2008)
OmpX
1, 2-di-cn-sn-glycero-phosphocholine
PNAS 98 (2002)
pSRII
NSMB. 17 (2010)
n=7 n=6
24 Å24 Å
25 Å24 Å 22 Å
22 Å
22 Å
24 Å30 Å
Beyond Hydrophobic Length
TM0026
DsbB
Lac permease
VDAC
Hydrophobic surface area and perturbation of micelle properties
Stability of tertiary fold
Stabilizing membrane proteins for structural and functional
studies
Investigating the structure and function of bacteria membrane
protein – host interactions
Developing quantitative spectroscopic methods for
investigating membrane protein structure and function
Biophysical methodsNMREPR
SAXSCrystallography
CEACAM and HSPG receptors
Curr Opin Microbiol 6, 43 (2003)Curr Opin Microbiol 6, 43 (2003)
CEACAM1 N-domainPredominant disaccharide
repeat in heparanActa Cryst. D Biol Crystallogr. 62, 971 (2006)
OpaHS and OpaCEA
What determines the receptor specificity of Opa to CEACAM versus HSPG?
• Determine the structure and dynamics of a variety of Opa proteins with and without receptor
• Determine specificity and affinities of Opa proteins
• Test the structural and dynamical models in vivo
OpaCEA
OpaHS
OpaCEA
OpaHS
OpaCEA
OpaHS
OpaCEA
OpaHS
In micellar and lipid environments…
Structure and dynamics of Opa with and without bound receptor.
- in micelles with NMR- in lipid vesicles with EPR
Affinities and specificities of Opa proteins for receptors
-in micelles with NMR- in lipids with fluorescence or EPR
NMR structure determination
Prepare stable sample
Record multidimensional spectra
Assign backbone atoms Assign sidechain atoms
Assign NOESY spectra(provide distance restraints)
Calculate structure
Proteins 60: 552-557 (2005)
Deuterated samples
TROSY pulse sequences
Specific sidechain labeling required
Limited NOE data:Additional restraintsinformation required
Nat. Struct. Bio. 8, 334 (2001)
Opa refolding in detergent micelles
OG DDM DM FC-12 s i s i s i s i
U
I
F
50 kDa
35
25
A B
Isolated inInclusion bodies
Resuspended in8M urea
Dilute indetergent
NMR of Opa proteins15N, 1H –TROSY NMR spectra
OpaCEA OpaHS
ω1 ( 15N
)[ppm]
107
113
119
125
131
9 8 10 9 8 7
Asp/Tyr Glu/Ile
ω2(1H)[ppm]10
107
113
119
125
131
107
113
119
125
131
ω1 ( 15N
)[ppm]
10 9 8 7ω2(1H)[ppm]
Assignment Strategy:Specific amino acid labeling
Lys/Val Arg/Ser
OpaCEA
kDa35
25
F +
Assignment Strategy: Trypsin digestionω
1 ( 15N)[ppm
]
ω2(1H)[ppm]7.08.09.010.0
106
110
114
118
122
126
130
MGSSHHHHHH SSGLVPRGSH MASEDGGRGP
YVQADLAYAY EHITHDYPEP TAPNKNKIST
VSDYFRNIRT RSVHPRVSVG YDFGGWRIAA
DYARYRKWNN NKYSVNIENV RIRKENGIRI
DRKTENQENG TFHAVSSLGL SAIYDFQIND
KFKPYIGARV AYGHVRHSID STKKTIEVTT
VPSNAPNGAV TTYNTDPKTQ NDYQSNSIRR
VGLGVIAGVG FDITPKLTLD AGYRYHNWGR
LENTRFKTHE ASLGVRYRF
Full lengthTrypsin digested
Trypsin
In micellar and lipid environments…
Structure and dynamics of Opa with and without bound receptor.
- in micelles with NMR- in lipid vesicles with EPR
Affinities and specificities of Opa proteins for receptors
-in micelles with NMR- in lipids with fluorescence
β-barrel membrane proteins can spontaneously refold into lipid bilayers
• If reversible, thermodynamics of folding can be measured.
• Function can be assessed in more physiological conditions
β-barrel folding into lipid bilayers assessed by ultracentrifugation and SDS-PAGE
β-barrel folding into lipid bilayers assessed by ultracentrifugation and SDS-PAGE
Fraction Folded =
Folded(Aggregate + Associated + Folded)
β-barrel folding into lipid bilayers assessed by ultracentrifugation and SDS-PAGE
Fraction Aggregate
=Aggregate
(Aggregate + Associated + Folded)
β-barrel folding into lipid bilayers assessed by ultracentrifugation and SDS-PAGE
Lipid Fraction Folded
=Folded
(Associated + Folded)
Opa proteins do not fold under conditions successful for OmpA…
OmpA folding conditions applied to Opa60
Ref. Optimized Folding Conditions Folding EfficiencyLipid,
Temperature (oC)Buffer,
pH OmpA Opa60
Surrey, 1992 diC14PC,30
10 mM KPi,7.3 40-50% <1%
Surrey, 1995diC14PC,
3020 mM
glycine, 10 >95% <1%
Kleinschmidt, 1996
DOPC,40
10 mM glycine, 150 mM NaCl,
1 mM EDTA, 8.3
>95% <1%
Hong, 2004
POPC or92.5/7.5
POPC/POPG,37.5
10 mM glycine, 2 mM
EDTA, 10>95% <1%
…nor into E. coli lipid extracts.
Total lipid extract is 57.5% PE, 15.1% PG, 9.8% cardiolipin, and 17.6% of uncharacterized mass.
Polar extract is 67.0% PE, 23.2% PG, 9.8% cardiolipin.
Lipid-chain length modulates Opa folding
Dewald et al. Biophys J in press
OpaCEA
Carbon chain length
Lipid chain length modulates membrane surface properties
van der Waalsattractive forces
electrostaticrepulsive forces
• Larger area per headgroup
• Increased [H2O] at bilayer surface• 0.5 M H2O /
2 carbons length
Shorter chain length or higher temperature:
Opa folding improves with basic pH
* folding not observed in at least three separate experiments; † observed value constant across replicates; ǂ folding seen in only one of three replicates, deviation not calculated.
C14PC
pI of OpaCEA ≈9.6
†
Positive membrane dipole potential may facilitate insertion of negatively charged Opa protein
Dipole Potential+ + + + 300 mV
Due to dipole potential, hydrophobic anions permeate a membrane 106 x faster than cations.
Protein charge prevents
aggregation
Opa proteins do not fold in ether-linked lipids.
Ether or ester linked diC14 PC
Opa concentration and molar lipid to protein ratio were 6 µM and 400.
The dipole potential of ether- linked DOPC is ~ 120 mV less than of ester-linked DOPC.
Summary of physical influences on Opa folding in lipid bilayers
Increasing…
Overall folding
efficiency AggregateLipid fraction
folded
pH
Ionic strength
Charged lipids
Unsaturated lipids
Urea N/A
Dewald et al. Biophys J in press
Liposomes containing Opa stimulate ROS production in human neutrophils
Luminol + ROS [3-APA*] 3-APA + light
Stabilizing membrane proteins for
structural and functional studies
Investigating the structure and
function of bacteria
membrane protein – host interactions
Developing quantitative
spectroscopic methods for investigating membrane
protein structure and function
Biophysical methodsNMREPR
SAXSCrystallography
The Columbus Group Ryan Alison Brett Dan Ryan Iza Oliver Dewald Kroncke Fox Lo Bielnicka
http://www.columbuslabs.org/ [email protected]
Support for this research provided by the NIH (RO1 GM087828-02), NSF (CAREER award MCB0845668), The Research Corporation, and the Jeffress Memorial Trust.
Ashley Justin Golda Tsiga Jackie Keller Kim Harris Solomon Hodges
Small Angle X-ray Scattering Jan Lipfert Sebastian Doniach
UVA MicrobiologyAlison CrissLouise Ball
Urea improves OpaI folding in diC10PC lipids
M- MW marker, U- unfolded control. Prespin samples are shown.
Dewald et al. Biophys J in press
Lipid-reconstituted Opa Proteins bind receptor
Opa & Opa, lipid, Lipid &lipid & CEACAM CEACAM or heparin or heparin
Fluorescence pull-down assay
Inte
nsity
at 3
10 n
m
1.8E7
9.0E6
Heparin - + + +2x +2xOpaHS + - + - +
Inte
nsity
at 3
10 n
m
1.0E6
5.0E5
CEACAM - + + +2x +2xOpaCEA + - + - +
Supernatant Fluorescence