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