1. 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

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

3. 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

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. 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.

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

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

8. Solubilization screens
Paul Wright and Michael Wiener, UVa

9. 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

10. Solubilization test of Rv0936-pstA2

11. Solubilization results

12. 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

13. 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

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

15. 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 °C.

16. 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

17. 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

18. Lyophilized samples
Natural abundant ubiquitin, overnight

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

20. 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