Membrane Protein Structural Genomics: A Multi-technology Challenge

University of Virginia

Yelena Peskova Kim DiGiandomenico Robert Nakamoto Paul Wright Michael Wiener

Florida State University and the National High Magnetic Field Laboratory

Alla Korepanova Philip Gao Yuanzhi Hua Tim Cross

Case Western Reserve University

Frank Soennichsen Kenneth Taylor

Vanderbilt University

Charles Sanders

Protein Structure Initiative of NIGMS-P01 GM64676

Holistic approach towards membrane protein structure

EXPRESSION & SAMPLE PREPARATION SOLUTION NMR SOLID STATE NMR X-STALLOGRAPHY ELECTRON MICROSCOPY STRUCTURE

7

M. tuberculosis

Membrane Protein Expression Results 100 80 60 40 20 160 140 120 100 80 60 40 20 0 0 <10 10-20 20-30 30-40 40-50 50-100 >100 Protein Mass (kDa) 1 2 3 4 # of Transmembrane Helices ≥ 5 T

Targeted Cloned Expressed

328 targets 228 cloned 150 express ~66% of clones express to some degree ~ 40 detected by Coomassie

Distribution of expressing proteins

16 14 12 10 8 6 4 2 0 0 20 40 60 Protein Mass (kDa) 80 0 <1 1 to 5 >5 100

Effect of tag position

40 30 20 10 0 N C Other observations: T7 promoter always works best C43 generally gives better expression levels

Solubilization screens

Paul Wright and Michael Wiener, UVa

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

Solubilization test of Rv0936-

pst

A2

Solubilization results

100nm

• Rv 0424c

(

hypothetical protein

) -12.7 kDa - pMCSG7/BL21 CodonPlus-(DE3)- RP • Electron Microscopy -JEM-1200EX -40k Mag.

-100kV

• Rv 2433c

• (

hypothetical protein

) • 11.3 kDa • pET-16b/BL21 CodonPlus-(DE3)-RP • Electron Microscopy • JEM-1200EX • 65k Mag • 100kV

More Tubular Structures from Rv 2433c

100nm 100nm

Quality – most informative TROSY-HSQC experiment DPC A B C D E F G H 1 H 15 N 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% D 2 O, pH=7.5, the spectra were measured at 35-45 °C.

The effect of detergent

DPC 45 º C Sarc 40 ºC LPPG 40 ºC (identical for LMPG, LOPG) Rv 1616 (conserved hypothetical protein), 15.3 kDa, 132 aa, 3 TM

Rv3368 • 1 TM, 214 aa • 22.3 kDa • (

conserved hypothetical protein

) • DPC • 800MHz • TROSY • 40 ºC • >150 peaks DPC (75%)

1TM, 214 aa, 22.3 kDa

Sarcosyl (>90%)

Lyophilized samples Natural abundant ubiquitin, overnight

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

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