There is an urgent need to develop new tools to facilitate crystallization of membrane proteins. The long-term goal of this application is to understand the molecular mechanisms of physiologically important and disease-related membrane proteins by X-ray crystallographic determination. The overall objective, which is an important step towards attainment of our long-term goal, is to test a novel approach -- the production and use of membrane protein capture reagents to facilitate crystallization and structure determination of membrane transporters. Although successful application of capture reagents as crystallization chaperones for structure determination of membrane proteins has been reported, the approach has not been widely utilized. We also suggest that novel molecules capable of trapping weakly bound but physiologically important membrane protein complexes in situ will be discovered. In this proposal, scaffold proteins-based combinatorial libraries will be constructed and tested by in vitro ribosome displays and in vivo functional screening to discover specific capture reagents that bind to the bacterial melibiose permease (MelB) or to the human red blood cell glucose transport (GLUT1). A proof-of-principle study on a capture reagent for trapping weakly interacting protein-membrane protein complexes will be carried out using well-characterized lactose permease (LacY) or MelB bound to their regulatory enzyme IIAglc. Specificity and affinity of selected capture reagents will be evaluated by biochemical and biophysical methods. Furthermore, selected capture reagents will also be tested for co-crystallization with their targeted membrane permeases. This will expedite the crystal structure determination of MelB. The impact of this proposal is that the methodology established will serve as a general tool for membrane protein structure determination, as well as functional studies, and the novel high-resolution structure of MelB will advance our knowledge of function at the atomic level. The proposed research is significant because the approach proposed addresses a critical barrier to progress in the field of membrane protein structure determination.

Public Health Relevance

Membrane proteins play crucial roles in many aspects of cell function and are the main targets of pharmacologically and toxicologically active substances. In comparison to soluble proteins, the number of available membrane protein structures is very limited, and this lack of high-resolution structures precludes deeper insights into essential aspects of human health and disease. The approach proposed here can serve as a general tool to enhance the probability of crystallization of membrane proteins and thereby facilitate their structure determination.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095538-03
Application #
8311706
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
2012-08-01
Budget End
2013-07-31
Support Year
3
Fiscal Year
2012
Total Cost
$297,000
Indirect Cost
$97,000
Name
Texas Tech University
Department
Physiology
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
Ethayathulla, Abdul S; Yousef, Mohammad S; Amin, Anowarul et al. (2014) Structure-based mechanism for Na(+)/melibiose symport by MelB. Nat Commun 5:3009
Amin, Anowarul; Ethayathulla, Abdul S; Guan, Lan (2014) Suppression of conformation-compromised mutants of Salmonella enterica serovar Typhimurium MelB. J Bacteriol 196:3134-9
Guan, Lan; Jakkula, S Vivek; Hodkoff, Alexey A et al. (2012) Role of Gly117 in the cation/melibiose symport of MelB of Salmonella typhimurium. Biochemistry 51:2950-7
Kudugunti, Shashi K; Thorsheim, Helen; Yousef, Mohammad S et al. (2011) The metabolic bioactivation of caffeic acid phenethyl ester (CAPE) mediated by tyrosinase selectively inhibits glutathione S-transferase. Chem Biol Interact 192:243-56
Guan, Lan; Nurva, Shailika; Ankeshwarapu, Siva P (2011) Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium. J Biol Chem 286:6367-74