The membrane proteome of T. maritima

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Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins for NMR structural studies

description

Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins for NMR structural studies. Functional Annotations of the Membrane Proteome. ABC transporters. 20%. 55%. Proteins with unknown function. 25%. Other functionally - PowerPoint PPT Presentation

Transcript of The membrane proteome of T. maritima

Page 1: 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

Page 2: The membrane proteome of  T. maritima

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

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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

Page 4: The membrane proteome of  T. maritima

# 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.

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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

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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

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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.

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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

Page 9: The membrane proteome of  T. maritima

Soluble ≠ folded!

9.5 9.0 8.5 8.0 7.5 7.0 6.5

1H (ppm)

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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.

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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.

Page 12: The membrane proteome of  T. maritima

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).