The membrane proteome of T. maritima
description
Transcript of The membrane proteome of T. maritima
Expression and purification of membrane proteins: Initial screening
of Thermotoga maritima α-helical membrane proteins for NMR
structural studies
The membrane proteome of T. maritima
24%
76%
Membrane proteins
Cytosolic proteins
Percentage of Membrane Proteins in T. maritima
Electron micrograph of T. maritima. The arrow points to the outer membrane, the “toga”, for which the organism is named.
55%
20%
25%Proteins with unknown function
Other functionally annotated proteins
ABC transportersFunctional Annotations of the Membrane Proteome
Target selection
Determine localization
Insoluble fraction Membrane fraction
Optimize expression to the membrane
Refold Extract
Co2+ or Ni2+-affinity purification
Detergent stability screen
NMR spectroscopy
Assay expression in E.coli
Overall Approach to Preparing Membrane Protein Samples for NMR Studies
# ofproteins
0 200 400 600 800 1000 1200 1400 1600
Protein length (amino acids)
Target Selection
Size distribution of the membrane proteome
NMR sample requirements600 L of 1 mM proteinuniform 15N and 13C-labelingmonodisperseMW < 30 kD (for standard NMR experiments, development
of TROSY has extended the MW limit for NMR studies)
Selected fifty targets less than 16 KD (~130 aa) that contained one to four predicted transmembrane segments.
Expression Assay
21.514.46.0
kD
Eleven out of fifty targets overexpressed using arabinose induction.
Par
a6-aa
Pml I
6-hisCAC GTG TM gene TTA ATT AA
Pac I
Pme I
Nco I
RBS Sma IPT
7
Expression Vector96 x 65 mL Fermentor
Localization of target membrane proteins
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.
IB S M IB S M
TM1554 TM1634
Soluble, monodisperse, and foldedIs 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.
Detergent Screen P S P S P S P S P S
OG NG DG OM DM
DoDM DHPC
P S P S P S P S P S
CHAPSLDAODPC
W indole side chain protons
1H (ppm)
Aromatic side chain protonsBackbone
amide protons
TM0361
Soluble ≠ folded!
9.5 9.0 8.5 8.0 7.5 7.0 6.5
1H (ppm)
110
115
120
125
130
110
115
120
125
11.0 10.0 9.0 8.0 7.0 9.0 8.08.5 7.5 7.01H (ppm)
15N (ppm)
1H (ppm)
15N (ppm)
TM0361 TM1634
130
105
Optimization of solution conditions for structure determination: 2D spectroscopy of 15N-labeled protein
Using the 96 x 65 mL fermentor, these samples were prepared with less than 1L of commercial media.
Summary
1. 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.
Acknowledgements
Kurt Wüthrich
Scott LesleyHeath KlockBernhard GeierstangerJoanna 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).