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

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

Membrane proteins are often thought to be very large proteins - indeed the membrane protein structures in the protein data bank suggest that in general these proteins are very large. Here the molecular weight distribution of the putative membrane proteins from TB is shown - 62% of the membrane proteins have a molecular weight less than 40kD, and more than 15% have a molecular weight less than 20 kDa Of course we must keep in mind that these monomer molecular weights do not reflect the molecular weights of the functional proteins which are typically oligomers or multi-protein complexes. In addition we display the number of proteins with a given number of transmembrane helices. Again, I am asked frequently how am I going to resolve the signals from seven transmembrane helices - well - only 16% of membrane proteins have such a large number of helices - at least in this genome. Note that there does not appear to be a correlation between these distributions. In fact there is very little correlation except that the relatively few proteins that have a large number of transmembrane helices are also quite large in molecular weight. 7

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

94% of these proteins have been expressed in inclusion bodies 53% have been expressed in membrane proteins 14% have been expressed in a soluble fraction - interestingly half of these were also expressed in the membrane fraction as well. As a function of the number of helices only 9% of the proteins with a single TM helix are expressed in the membrane fraction while more than 80% of the proteins having three or more helices are expressed in the membrane fraction.

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

Effect of tag position N C # Expressing Proteins 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-pstA2

Solubilization results

Rv 0424c (hypothetical protein) 100nm Rv 0424c (hypothetical protein) -12.7 kDa - pMCSG7/BL21- CodonPlus-(DE3)- RP Electron Microscopy -JEM-1200EX -40k Mag. -100kV See tubes in 3 of 21 protein overexpression strains

Rv 2433c Electron Microscopy 11.3 kDa pET-16b/BL21-CodonPlus-(DE3)-RP (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 B C D E F G H A Rapid spectra: usually less than expected peaks: Resolution, peak overlap, variablility in linewidths, sensitivity Criteria: Peak dispersion,Peak overlap,Peak number Variability in linewidths 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.

The effect of detergent About 125 peaks in Sarcosyl, about 93 (clear) in LPPG about half in DPC Differences in resonance frequencies could be (although not necessarily) suggestive of different conformation/structure in these different detergents. For this example, this is supported by CD differences between DPC and LPPG (by about ? %) 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

Rv3368 DPC (75%) Sarcosyl (>90%) 1 TM, 214 aa 22.3 kDa (conserved hypothetical protein) DPC 800MHz TROSY 40 ºC >150 peaks 25 kDa, 1 TM 1TM, 214 aa22.3 kDa DPC (75%) Sarcosyl (>90%) 1TM, 214 aa, 22.3 kDa

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