Crystallization of membrane proteins in lipidic mesophases (in meso) is a promising technique, which recently yielded high-resolution structures of human ?2-adrenergic and adenosine A2A G protein-coupled receptors. A distinct feature of the in meso crystallization is that the protein of interest is embedded into a highly curved and folded in space lipid bilayer, forming a lipidic cubic phase. The structure of the cubic phase imposes spatial constrains on diffusion of large proteins or oligomeric protein aggregates. Our preliminary data indicate that one of the primary reasons for failure of the in meso crystallization trials is due to a fast nonspecific protein aggregation. There is no visual feedback to such event, the cubic phase remains clear, because the size of the non-diffusing stuck protein oligomeric aggregates is well below 100 nm. The aggregation behavior of a protein depends on the particular protein construct, host lipid and additives employed for crystallization. We propose to develop an automated system for carrying out Fluorescence Recovery after Photobleaching (FRAP) studies of membrane proteins in meso. This system will be used to develop a pre-screening assay which will use less than 10 ?L of fluorescently labeled protein solution to screen for diffusion of the protein in the lipidic cubic phase incubated with 96 solutions of carefully selected common precipitants. The results of this assay will help to select suitable for in meso crystallization protein constructs and to eliminate certain precipitants from subsequent crystallization trials. Such crystallization pre-screening approach will significantly increase chances of obtaining initial crystal hit leading to the determination of more high-resolution structures of challenging membrane proteins essential in designing new and improved highly specific drugs with lesser side effects. PUBLIC HEALTH RELEVENCE (provided by the applicant): This research project will develop new technologies that will accelerate the success rate of membrane protein crystallization. These proteins are involved in important cellular and physiological processes and therefore are important drug targets. Crystallization will facilitate solution of three-dimensional structures of the proteins providing necessary templates for the rational drug development process.

Public Health Relevance

This research project will develop new technologies that will accelerate the success rate of membrane protein crystallization. These proteins are involved in important cellular and physiological processes and therefore are important drug targets. Crystallization will facilitate solution of three-dimensional structures of the proteins providing necessary templates for the rational drug development process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory/Developmental Grants (R21)
Project #
8R21GM103972-03
Application #
8197583
Study Section
Special Emphasis Panel (ZRR1-BT-7 (01))
Program Officer
Friedman, Fred K
Project Start
2010-01-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2013-11-30
Support Year
3
Fiscal Year
2012
Total Cost
$235,001
Indirect Cost
$111,251
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037