The purpose of the proposed research is to develop a set of NMR strategies for membrane protein structure determination. Combined with our existing E. coli-based cell-free (CF) expression system, these strategies will then be used to solve several structures of human integral membrane proteins (hIMPs). The existing NMR structure determination methods are not effective for membrane proteins because of their intrinsic structural and dynamical properties. The internal mobility of transmembrane (TM) helical bundles causes strong broadening of the signals in NMR spectra and creates problems with signal assignment, spectra analysis, and detection of long-range interactions, which are necessary to build up the structure of the TM 1-helical domain. Therefore, we propose a set of NMR strategies utilizing the CF expression system, which will significantly speed up structure determination of membrane proteins. In particular, we will further develop the combinatorial dual-isotope labeling strategy in order to make the resonance assignment process fast and robust (Aim 1);incorporate unnatural amino acids that can be modified with a paramagnetic label to measure relaxation enhancement and paramagnetic chemical shift effects (Aim 2);implement 13C-methyl and 19F-labeling in the CF system to obtain additional long-range distance constraints for structure determination (Aim 3). These methods will be applied to preselected (Aim4) hIMPs to determine the high resolution NMR structures of 10 or more targets (Aim 5).

Public Health Relevance

In the proposed study we aim to create innovative tools for structure determination of membrane proteins and use them to solve several human integral membrane protein structures. 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 the proposed aims, we are going to utilize a novel in vitro expression system to modify the existing and create new high-speed NMR methods for the determination of membrane protein structures.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098630-04
Application #
8705542
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Wehrle, Janna P
Project Start
2011-08-15
Project End
2015-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
4
Fiscal Year
2014
Total Cost
$363,044
Indirect Cost
$163,044
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