This proposal addresses challenges in eukaryotic protein expression, solublization, stablization and crystallization. We will accomplish this by integrating early assessment measurements of protein quality and quantity into an existing robust mammalian cell expression platform. This approach integrates use of a novel high-throughput self-interaction chromatography system (HT-SIC) that rapidly measures second virial coefficients for the membrane protein mixed with a specially designed panel of additives. An artificial neural network analyzes the experimentally derived second virial coefficient data and performs in silico predictions of novel solution conditions that improve protein solubility and homogeneity. The HT-SIC system also enables informed adjustments to solution conditions that alter protein-protein interactions such that the probability of producing high-quality crystals is improved. Achieving the specific aims of this proposal will provide the research community with significant advancements toward a cost effective, knowledge-based approach to express, purify, stabilize and crystallize membrane proteins. Target proteins include two ion channel proteins (epithelial sodium channel, ENaC and cystic fibrosis transmembrane regulator protein, CFTR), two GPCRs (chemokine receptor-1, CCR1 and sphingosine- 1 phosphate receptor, S1P) and growth hormone receptor, GHR. Structures of these proteins would contribute significantly to our understanding of their biological mechanism of action and role in several important diseases including cancer (colon, breast and prostate), diabetes, cystic fibrosis, growth anomalies, immune system disorders, hypertension, sepsis and the flu. For each IMP, we have established collaborations with biochemists/biologists with a long-standing interest in and experience working with each target protein and its protein interactome.

Public Health Relevance

This proposal is directed at the development of novel protocols to produce, purify, stabilize and crystallize integral membrane proteins. This will be accomplished using several innovative approaches and technologies. The proteins targeted are directly associated with human disease including cancer (colon, breast and prostate), diabetes, cystic fibrosis, growth anomalies, immune system disorders, hypertension, sepsis and the flu.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095639-04
Application #
8520338
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
$288,090
Indirect Cost
$91,442
Name
University of Alabama Birmingham
Department
Other Health Professions
Type
Schools of Optometry/Ophthalmol
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Wilson, William W; Delucas, Lawrence J (2014) Applications of the second virial coefficient: protein crystallization and solubility. Acta Crystallogr F Struct Biol Commun 70:543-54
Johnson, David H; Wilson, W William; DeLucas, Lawrence J (2014) Protein solubilization: a novel approach. J Chromatogr B Analyt Technol Biomed Life Sci 971:99-106
Hildebrandt, Ellen; Zhang, Qinghai; Cant, Natasha et al. (2014) A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR). Biochim Biophys Acta 1838:2825-37
Lazrak, Ahmed; Fu, Lianwu; Bali, Vedrana et al. (2013) The silent codon change I507-ATC->ATT contributes to the severity of the ?F508 CFTR channel dysfunction. FASEB J 27:4630-45