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University of Virginia Yelena Peskova Kim DiGiandomenico Robert Nakamoto Paul Wright

Membrane Protein Structural Genomics: A Multi-technology Challenge. Florida State University and the National High Magnetic Field Laboratory Alla Korepanova Philip Gao Yuanzhi Hua Tim Cross Kenneth Taylor. University of Virginia Yelena Peskova Kim DiGiandomenico Robert Nakamoto

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University of Virginia Yelena Peskova Kim DiGiandomenico Robert Nakamoto Paul Wright

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  1. Membrane Protein Structural Genomics: A Multi-technology Challenge Florida State University and the National High Magnetic Field Laboratory Alla Korepanova Philip Gao Yuanzhi Hua Tim Cross Kenneth Taylor University of Virginia Yelena Peskova Kim DiGiandomenico Robert Nakamoto Paul Wright Michael Wiener Case Western Reserve University Frank Soennichsen Vanderbilt University Charles Sanders Protein Structure Initiative of NIGMS-P01 GM64676

  2. Holistic approach towards membrane protein structure EXPRESSION & SAMPLE PREPARATION SOLUTION NMR SOLID STATE NMR ELECTRON MICROSCOPY X-STALLOGRAPHY STRUCTURE

  3. 7

  4. M. tuberculosis Membrane Protein Expression Results # of Proteins Targeted Cloned Expressed <10 10-20 20-30 30-40 40-50 50-100 >100 Protein Mass (kDa) 328 targets 228 cloned 150 express ~66% of clones express to some degree ~ 40 detected by Coomassie # of Proteins 1 2 3 4 ≥5 # of Transmembrane Helices

  5. Distribution of expressing proteins # of Transmembrane Helices Protein Mass (kDa)

  6. Effect of tag position # Expressing Proteins N C Other observations: T7 promoter always works best C43 generally gives better expression levels

  7. Solubilization screens Paul Wright and Michael Wiener, UVa

  8. Solubilization screen procedure Membranes are incubated with detergent at 10x CMC for 1 hr at RT Suspension is centrifuged for 1 hr at 155,000 g SDS-PAGE of pellet and s/n, visualized by immunoblot

  9. Solubilization test of Rv0936-pstA2

  10. Solubilization results

  11. 100nm • Rv 0424c (hypothetical protein) -12.7 kDa - pMCSG7/BL21- CodonPlus-(DE3)- RP • Electron Microscopy -JEM-1200EX -40k Mag. -100kV

  12. 100nm 100nm • Rv 2433c • (hypothetical protein) • 11.3 kDa • pET-16b/BL21-CodonPlus-(DE3)-RP • Electron Microscopy • JEM-1200EX • 65k Mag • 100kV

  13. More Tubular Structures from Rv 2433c 100nm 100nm

  14. A B C D E F G H Quality – most informative TROSY-HSQC experiment DPC 1H-15N TROSY spectra of Rv0011c (A), Rv1342c (B), Rv2199c(C), Rv3782(D), Rv1616(E), Rv3368c (F), Rv3773c (G), Rv2599(H) in 5% DPC, 250mM Imidazole, 10% D2O, pH=7.5, the spectra were measured at 35-45 °C.

  15. The effect of detergent DPC 45 ºC Sarc 40 ºC LPPG 40 ºC (identical for LMPG, LOPG) Rv 1616 (conserved hypothetical protein), 15.3 kDa, 132 aa, 3 TM

  16. Rv3368 • 1 TM, 214 aa • 22.3 kDa • (conserved hypothetical protein) • DPC • 800MHz • TROSY • 40 ºC • >150 peaks DPC (75%) Sarcosyl (>90%) 1TM, 214 aa, 22.3 kDa

  17. Lyophilized samples Natural abundant ubiquitin, overnight

  18. Crystallized proteins natural abundant 14 hours (2.5s/scan)

  19. Summary • Success rate for expression of IMP is about the same as for soluble proteins but levels are much lower • Many smaller proteins with 1-3 TMs express into inclusion bodies – good over-expression but proteins must be refolded • Initial NMR spectra suggest that aggregated proteins can be refolded • Important to test expression with tags at either end of protein • Important to screen through many detergents • Over-expression of some membrane proteins create intracellular membrane tubes • Solid state NMR of micro-crystals is promising approach

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