Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins

1. Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins for NMR structural studies

2. Functional Annotations of the Membrane Proteome ABC transporters 20% 55% Proteins with unknown function 25% Other functionally annotated proteins Membrane proteins 24% 76% Cytosolic proteins The membrane proteome of T. maritima Electron micrograph of T. maritima. The arrow points to the outer membrane, the “toga”, for which the organism is named. Percentage of Membrane Proteins in T. maritima

3. Overall Approach to Preparing Membrane Protein Samples for NMR Studies

4. # of proteins 0 200 400 600 800 1000 1200 1400 1600 Protein length (amino acids) Target Selection NMR sample requirements 600 mL of 1 mM protein uniform 15N and 13C-labeling monodisperse MW < 30 kD (for standard NMR experiments, development of TROSY has extended the MW limit for NMR studies) Size distribution of the membrane proteome Selected fifty targets less than 16 KD (~130 aa) that contained one to four predicted transmembrane segments.

5. Para PT7 Sma I Pme I RBS 6-aa 6-his CAC GTG TM gene TTA ATT AA Nco I Pml I Pac I kD 21.5 14.4 6.0 Expression Assay 96 x 65 mL Fermentor Expression Vector Eleven out of fifty targets overexpressed using arabinose induction.

6. Localization of target membrane proteins TM1554 TM1634 IB S M IB S M In order to verify that the target proteins are membrane proteins, the E. coli membranes were isolated using ultracentrifugation and extracted with n-decyl--D-maltoside . Of the eleven expressing targets, two expressed exclusively in the insoluble fraction (IB) and nine expressed to both the insoluble fraction and the n-decyl--D-maltoside solubilized membrane (M) fraction. None of the proteins were found in the soluble (S) fraction.

7. Soluble, monodisperse, and folded Is that too much to ask? Which detergent will do all this? For now, there is no paradigm. There isn’t one detergent that works for all membrane proteins; therefore, for each protein, many detergents need to be screened.

8. Detergent Screen NG DG OM DM OG P S P S P S P S P S TM0361 Aromatic side chain protons Backbone amide protons DPC LDAO DoDM DHPC CHAPS W indole side chain protons P S P S P S P S P S 1H (ppm)

9. 9.5 9.0 8.5 8.0 7.5 7.0 6.5 Soluble ≠ folded! 1H (ppm)

10. Optimization of solution conditions for structure determination: 2D spectroscopy of 15N-labeled protein 9.0 8.5 8.0 7.5 7.0 11.0 10.0 9.0 8.0 7.0 105 TM0361 TM1634 110 110 115 115 15N (ppm) 120 15N (ppm) 120 125 125 130 130 1H (ppm) 1H (ppm) Using the 96 x 65 mL fermentor, these samples were prepared with less than 1L of commercial media.

11. Summary • Approximately 20% of the membrane protein targets express in HK100 E. coli cells with arabinose induction. • 2. Detergent screening has been successful in preparing solubilized membrane protein samples, but will be revised as we gain experience. • 3. Similar to soluble proteins, 1H 1D NMR spectroscopy is suitable for evaluating the overall fold of the protein. • 4. TM1634 has been optimized and NMR structure determination is in progress.

12. Acknowledgements Kurt Wüthrich Scott Lesley Heath Klock Bernhard Geierstanger Joanna Hale This work is funded by the NIH protein structure initiative grant P50 GM62411. LC is funded by NIH grant 1F32GM068286. KW is funded by the endowment of Cecil H. and Ida M. Green (TSRI).