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/ columbus@virginia.edu

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