This application focuses on upgrading the E. coli-based cell-free expression system and on optimizing the synergy between cell-free and NMR to screen and evaluate for structural studies by NMR and X-ray methods a library of 3360 human integral membrane proteins. Currently there exists an enormous knowledge gap for membrane protein structures. Fewer than 20 human membrane protein structures are available in the Protein Data Bank. Membrane proteins mediate cellular interactions with the surrounding, and they are targeted by half of the commercially available drugs. Each new human membrane protein structure is, therefore, a potential target of rational structure-guided drug design, and the significance of new structures cannot be overstated. We propose to improve the existing CF expression system optimized in our lab for membrane proteins to enable proper disulfide bridge formation and a large-size protein expression, and to improve purification of CF-expressed targets designated for crystallization screening (Aim1). We will also optimize the NMR structure determination method for membrane proteins utilizing the synergy between CF and NMR. We will improve the combinatorial dual-isotope labeling (CDL) strategy developed in our lab and use CF to incorporate site-specific unnatural amino acids. We will also employ 19F and 13C-methyl labeling (Aim 2). We will use these technical improvements to comprehensively evaluate the proposed human membrane protein targets by testing their expression and their suitability for NMR and X- ray structural studies (Aim3).

Public Health Relevance

In this proposed study we aim to create innovative tools for expression and subsequent structure determination of membrane proteins and use these tools to screen half of the human membrane proteome. Each human membrane protein structure is a potential target for rational structure-guided drug design and the significance of new structures cannot be overstated. To achieve proposed aims we will modify and upgrade a novel in vitro expression system to produce human integral membrane proteins and combine it with high resolution NMR techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095623-04
Application #
8517748
Study Section
Special Emphasis Panel (ZRG1-BCMB-S (50))
Program Officer
Preusch, Peter C
Project Start
2010-09-30
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2013
Total Cost
$286,606
Indirect Cost
$136,157
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
078731668
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Lindert, Steffen; Maslennikov, Innokentiy; Chiu, Ellis J C et al. (2014) Drug screening strategy for human membrane proteins: from NMR protein backbone structure to in silica- and NMR-screened hits. Biochem Biophys Res Commun 445:724-33
Maslennikov, Innokentiy; Choe, Senyon (2013) Advances in NMR structures of integral membrane proteins. Curr Opin Struct Biol 23:555-62