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
Florida State University and
the National High Magnetic
Field Laboratory
Alla Korepanova
Philip Gao
Yuanzhi Hua
Tim Cross
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
STRUCTURE
ELECTRON
MICROSCOPY
7
M. tuberculosis Membrane Protein Expression Results
# of Proteins
100
80
T
Targeted
Cloned
Expressed
60
40
20
0
<10
10-20 20-30 30-40 40-50 50-100 >100
Protein Mass (kDa)
328 targets
228 cloned
150 express
160
# of Proteins
140
120
100
~66% of clones
express to some degree
~ 40 detected by
Coomassie
80
60
40
20
0
1
2
3
4
# of Transmembrane Helices
≥5
Distribution of expressing proteins
16
# of Transmembrane Helices
14
12
10
8
6
0
<1
1 to 5
>5
4
2
0
0
20
40
60
Protein Mass (kDa)
80
100
# Expressing Proteins
Effect of tag position
40
30
20
N
C
10
0
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
100nm
• Rv 0424c
(hypothetical protein)
-12.7 kDa
- pMCSG7/BL21CodonPlus-(DE3)- RP
• Electron
Microscopy
-JEM-1200EX
-40k Mag.
-100kV
100nm
100nm
• Rv 2433c
• (hypothetical protein)
• 11.3 kDa
• pET-16b/BL21CodonPlus-(DE3)-RP
• Electron Microscopy
• JEM-1200EX
• 65k Mag
• 100kV
More Tubular Structures from Rv 2433c
100nm
100nm
Quality – most informative TROSY-HSQC experiment
DPC
1H-15N
A
B
C
D
E
F
G
H
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
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
• 1 TM, 214 aa
• 22.3 kDa
• (conserved
hypothetical
protein)
•
•
•
•
DPC
800MHz
TROSY
40 ºC
• >150 peaks
DPC
1TM, 214 aa, 22.3 kDa
(75%)
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