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
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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
The membrane proteome of T. maritima
Electron micrograph of T. maritima. The arrow points to the outer membrane, the “toga”, for which the organism is named.
Percentage of Membrane Proteins in T. maritima
Overall Approach to Preparing Membrane Protein
Samples for NMR Studies
0 200 400 600 800 1000 1200 1400 1600
Protein length (amino acids)
NMR sample requirements
600 mL of 1 mM protein
uniform 15N and 13C-labeling
MW < 30 kD (for standard NMR experiments, development of TROSY has extended the MW limit for NMR studies)
Size distribution of the membrane proteome
Selected fifty targets less than 16 KD (~130 aa) that contained one to four predicted transmembrane segments.
CAC GTG TM gene TTA ATT AA
96 x 65 mL Fermentor
Eleven out of fifty targets overexpressed using arabinose induction.
Localization of target membrane proteins
IB S M
IB S M
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.
Soluble, monodisperse, and folded
Is 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.
P S P S P S P S P S
W indole side
P S P S P S P S P S
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.
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).