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Impact of protein – detergent interactions on NMR structure determination

Impact of protein – detergent interactions on NMR structure determination. Linda Columbus Department of Chemistry University of Virginia. Membrane Proteins. BtuB. LamB. OmpX. OM. KcsA. SecYEG. BtuCD. CM. < 100 polytopic membrane protein structures deposited in the pdb

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Impact of protein – detergent interactions on NMR structure determination

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

  2. Membrane Proteins BtuB LamB OmpX OM KcsA SecYEG BtuCD CM < 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 OmpG PagP dodecylphosphocholine octyl glucoside dodecylphosphocholine OmpA OmpX 1, 2-dihexanoyl-sn-glycero-phosphocholine dodecylphosphocholine

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

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

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

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

  8. Properties of detergent micelles hydrophobic tail polar head group monomer micelle • 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...

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

  10. Detergents influence the completeness of NMR observations and tertiary interactions • What process in DDM and FC-10 is contributing to line broadening? DM DDM 2. Why do DM and FC-12 PDCs have reduced line broadening? FC-10 FC-12

  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. Geometry of micelle influences the completeness of NMR observations and tertiary interactions 104 104 108 108 FC-10 DDM 112 112 116 116 15N (ppm) 120 120 124 124 128 128 9 8 7 9 8 7 1H (ppm)

  15. Predicted a DHPC/LPPG mixture based on the detergent properties. 106 110 114 15N (ppm) 118 122 126 9 8 7 1H (ppm) Hot off the spectrometer… Dan Fox and Kalyani Jambunathan

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

  17. Acknowledgements • Small Angle X-ray Scattering • Jan Lipfert • Sebastian Doniach 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

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