University of Virginia Yelena Peskova Kim DiGiandomenico Robert Nakamoto Paul Wright
description
Transcript of University of Virginia Yelena Peskova Kim DiGiandomenico Robert Nakamoto Paul Wright
University of Virginia
Yelena PeskovaKim DiGiandomenicoRobert Nakamoto
Paul WrightMichael Wiener
Case Western Reserve UniversityFrank Soennichsen
Vanderbilt UniversityCharles Sanders
Membrane Protein Structural Genomics:A Multi-technology Challenge
Florida State University and the National High Magnetic Field Laboratory
Alla KorepanovaPhilip GaoYuanzhi HuaTim Cross
Kenneth Taylor
Protein Structure Initiative of NIGMS-P01 GM64676
Holistic approach towards membrane protein structure
EXPRESSION &SAMPLE PREPARATION
SOLID STATENMR
SOLUTIONNMR
ELECTRONMICROSCOPY
X-STALLOGRAPHY
STRUCTURE
7
TargetedClonedExpressed
0
20
40
60
80
100
T
0
20
40
60
80
100
120
140
160
M. tuberculosis Membrane Protein Expression Results#
of
Prot
eins
# of
Pr
otei
ns
<10 20-30 30-4010-20 40-50 50-100 >100
Protein Mass (kDa)
1 32 ≥54# of Transmembrane Helices
328 targets228 cloned150 express
~66% of clones express to some degree ~ 40 detected by Coomassie
Distribution of expressing proteins
0
2
4
6
8
10
12
14
16
0 20 40 60 80 100
0
<1
1 to 5
>5
# of
Tra
nsm
embr
ane
Hel
ices
Protein Mass (kDa)
0
10
20
30
40
Effect of tag position
N C
# E
xpre
ssin
g P
rote
ins
Other observations:T7 promoter always works bestC43 generally gives better expression levels
Solubilization screensPaul Wright and Michael Wiener, UVa
Membranes are incubated with detergent at 10x CMC for 1 hr at RTSuspension is centrifuged for 1 hr at 155,000 g
SDS-PAGE of pellet and s/n, visualized by immunoblot
Solubilization screen procedure
Solubilization test of Rv0936-pstA2
Solubilization results
• Rv 0424c (hypothetical protein)
-12.7 kDa
- pMCSG7/BL21- CodonPlus-(DE3)- RP
• Electron Microscopy
-JEM-1200EX
-40k Mag.
-100kV
100nm
• Rv 2433c• (hypothetical protein)
• 11.3 kDa• pET-16b/BL21-
CodonPlus-(DE3)-RP
• Electron Microscopy• JEM-1200EX• 65k Mag• 100kV
100nm100nm
More Tubular Structures from Rv 2433c
100nm
100nm
Quality – most informative TROSY-HSQC experiment
B C D
E F G H
A
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.
DPC
The effect of detergent
Sarc 40 ºC LPPG 40 ºC
(identical for LMPG, LOPG)
DPC 45 ºC
Rv 1616 (conserved hypothetical protein), 15.3 kDa, 132 aa, 3 TM
Rv3368
DPC (75%) Sarcosyl (>90%)
1TM, 214 aa, 22.3 kDa
• 1 TM, 214 aa
• 22.3 kDa
• (conserved hypothetical protein)
• DPC
• 800MHz
• TROSY
• 40 ºC
• >150 peaks
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