Impact of protein – detergent interactions on NMR structure

determination

Linda ColumbusDepartment of Chemistry

University of Virginia

Membrane Proteins

OM

CM

BtuB LamB OmpX

KcsA SecYEG BtuCD

< 100 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

NMR membrane protein structures

dodecylphosphocholine

OmpG PagP

dodecylphosphocholine octyl glucoside

OmpX

1, 2-dihexanoyl-sn-glycero-phosphocholine

OmpA

dodecylphosphocholine

The role of detergents in membrane protein structural and functional studies

The role of detergents in membrane protein structural and functional studies

The role of detergents in membrane protein structural and functional studies

The role of detergents in membrane protein structural and functional studies

Properties of detergent micelles• Critical micelle concentration (cmc)

– Concentration of monomer in equilibrium with micelle• Aggregation number

– Number of monomers in micelle• Shapes and sizes – prolate, oblate, cylinder, etc...

monomer micelle

hydrophobic tail

polar head group

Model System: TM0026

15N (p

pm

)

1H (ppm)

N

C

Preliminary structural model

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?

What happens in FC-10 and DDM that contributes to line broadening?

SDSL indicates the helical interaction is disrupted in FC-10

Why do DM and FC-12 give rise to quality NMR spectra?

15N

(ppm

)

DDM104

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 ofNMR observations and tertiary interactions

Hot off the spectrometer…

Dan Fox and Kalyani Jambunathan

Predicted a DHPC/LPPG mixture based on the detergent properties.

15N

(ppm

)

106

110

114

118

122

126

9 8 71H (ppm)

Detergent (abbreviation) N Ionic property

n-octyl--D-glucopyranoside (OG) 75-100 Non-ionic

n-nonyl--D-glucopyranoside (NG) 160-180 Non-ionic

n-decyl--D-glucopyranoside (DG) ND Non-ionic

n-decyl--D-maltoside (DM) 70-75 Non-ionic

n-dodecyl--D-maltoside (DoDM) 130-140 Non-ionic

1,2-dihexanoyl-sn-glycero-phosphocholine (DHPC)

35-40 zwitterionic

3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS)

14-18 zwitterionic

Lauryldimethylyamine oxide (LDAO) NC zwitterionic

n-decylphosphocholine (FC-10) 35-45 zwitterionic

n-dodecylphosphocholine (FC-12) 65-70 zwitterionic

1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG)

100-105 ionic

Unique structure is stabilized by a hydrophobic match to the detergent shape and size

DM, FC-12, and FC-10/DoDM

DoDM

FC-10

Small Angle X-ray Scattering Jan Lipfert Sebastian Doniach

Acknowledgements Scott Lesley Gerard Kroon Bernhard Geierstanger

Kalyani Bill Brett DanJambunathan Peairs Kroncke Fox

Iza Thien Chris Justin Bielnicka Nguyen Reyes Kim

NRSA 1F32GM068286UVA Start-up funds