Impact of protein – detergent interactions on NMR structure determination Linda Columbus Department of Chemistry University of Virginia
Feb 06, 2016
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