1. Impact of protein – detergent interactions on NMR structure determination Linda Columbus Department of Chemistry University of Virginia

2. Membrane Proteins OM CM BtuBLamBOmpX KcsA SecYEGBtuCD < 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

3. NMR membrane protein structures dodecylphosphocholine OmpG PagP dodecylphosphocholine octyl glucoside OmpX 1, 2-dihexanoyl-sn-glycero-phosphocholine OmpA dodecylphosphocholine

4. The role of detergents in membrane protein structural and functional studies

8. 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... monomermicelle hydrophobic tail polar head group

9. Model System: TM0026 15 N (ppm) 1 H (ppm) N C Preliminary structural model

10. 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?

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

12. SDSL indicates the helical interaction is disrupted in FC-10

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

14. 15 N (ppm) DDM 104 108 112 116 120 124 128 987 1 H (ppm) FC-10 987 104 108 112 116 120 124 128 Detergent (abbreviation) Ag #L (Å) Shperoidr HC (Å) V HC (nm 3 ) Ionic property n-decyl-  -D-maltoside (DM) 80-9034oblate12, 2326Non-ionic n-dodecyl-  -D-maltoside (DDM) 135-14539oblate14, 2949Non-ionic n-decylphosphocholine (FC-10)45-5328prolate21, 1315zwitterionic n-dodecylphosphocholine (FC-12)65-8034prolate26, 1627zwitterionic Detergent (abbreviation)Ag #L (Å) Shperoidr HC (Å) V HC (nm 3 ) Ionic property n-decyl-  -D-maltoside (DM) 80-9034oblate12, 2326Non-ionic n-dodecyl-  -D-maltoside(DDM) 135-14539oblate14, 2949Non-ionic n-decylphosphocholine (FC-10)45-5328prolate21, 1315zwitterionic n-dodecylphosphocholine (FC-12)65-8034prolate26, 1627zwitterionic DDM/FC-10 (molar ratio 4:7) 77 (30/47) 34oblate14, 2126zwitterionic Geometry of micelle influences the completeness of NMR observations and tertiary interactions

15. Hot off the spectrometer… Dan Fox and Kalyani Jambunathan Predicted a DHPC/LPPG mixture based on the detergent properties. 15 N (ppm) 106 110 114 118 122 126 987 1 H (ppm) Detergent (abbreviation) NIonic property n-octyl-  -D-glucopyranoside (OG) 75-100Non-ionic n-nonyl-  -D-glucopyranoside (NG) 160-180Non-ionic n-decyl-  -D-glucopyranoside (DG) NDNon-ionic n-decyl-  -D-maltoside (DM) 70-75Non-ionic n-dodecyl-  -D-maltoside (DoDM) 130-140Non-ionic 1,2-dihexanoyl-sn-glycero- phosphocholine (DHPC) 35-40zwitterionic 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate (CHAPS) 14-18zwitterionic Lauryldimethylyamine oxide (LDAO)NCzwitterionic n-decylphosphocholine (FC-10)35-45zwitterionic n-dodecylphosphocholine (FC-12)65-70zwitterionic 1-palmitoyl-2-hydroxy-sn-glycero-3- [phospho-rac-(1-glycerol)] (LPPG) 100-105ionic

16. Unique structure is stabilized by a hydrophobic match to the detergent shape and size DM, FC-12, and FC-10/DoDM DoDM FC-10

17. Small Angle X-ray Scattering Jan Lipfert Sebastian Doniach Acknowledgements Scott Lesley Gerard Kroon Bernhard Geierstanger Kalyani Bill Brett Dan Jambunathan Peairs Kroncke Fox Iza Thien Chris Justin Bielnicka Nguyen Reyes Kim NRSA 1F32GM068286 UVA Start-up funds