Transmembrane proteins (TMPs) play important physiological roles, are the targets of most clinically useful drugs, and constitute 20-30% of most genomes, however they remain poorly characterized at the structural level. High-resolution structures are available for only ten unrelated eukaryotic TMPs (excluding mitochondrial and chloroplast proteins.) Two major barriers to determining structures of eukaryotic TMPs are the difficulty of achieving high levels of expression of TMPs in native conformations and the apparent instability of TMPs leading to heterogeneity and loss of native conformations during expression, purification, and crystallization for x-ray diffraction. This application describes a set of genetic procedures for overcoming these barriers focusing on yeast as a genetically tractable organism that is the expression host with the most successful history of production of eukaryotic TMPs for x-ray crystallography. The overall approach will be to use flow cytometry in conjunction with fluorescent ligands that bind specifically to native conformations of TMPs to screen large libraries of mutated and otherwise modified yeast cells to identify alterations that enhance the levels of expression and stabilities of TMPs. These procedures will initially be applied to G protein Coupled Receptors (GPCRs), including mammalian GPCRs that can be expressed in yeast and the endogenous yeast receptor for 1-mating pheromone. Both the procedures and the high-expressing strains developed by the project are expected to be applicable to other classes of TMPs. Screening based on binding of fluorescent ligands will be used to: 1) Identify alterations in yeast host strains leading to increased expression of functional receptors. Some of these alterations will be introduced using a new approach, global transcription machinery engineering that provides the ability to alter expression of multiple genes at the same time. Other alterations will be effected using strains derived from yeast genomic deletion collection and by transforming receptor-expressing strains with libraries of overexpressed yeast genes and mammalian cDNAs;2) Identify mutations in the genes encoding GPCRs that lead to increased expression;and 3) Identify mutations that render GPCRs resistant to thermal denaturation. As high-level expression of stabilized GPCRs expressed in yeast is achieved, the receptors will be purified, tested for stability in detergent solutions, and subjected to crystallization trials for structure determination.

Public Health Relevance

This project focuses on the development of genetic approaches to aid in determination of the structures of transmembrane proteins. Given the small number of known structures of eukaryotic membrane proteins, and their diverse medical relevance, providing methods to solve even a few such structures could have a large impact on understanding of the functions of such proteins and in the design of drugs to alter their behaviors. The new approaches will be applied initially to structure determination of G protein coupled receptors, which are targets of a large fraction of clinically useful drugs and play critical roles in a wide variety of signaling pathways.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM084083-01A1
Application #
7583263
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Edmonds, Charles G
Project Start
2009-01-01
Project End
2012-12-31
Budget Start
2009-01-01
Budget End
2009-12-31
Support Year
1
Fiscal Year
2009
Total Cost
$334,709
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Sridharan, Rajashri; Connelly, Sara M; Naider, Fred et al. (2016) Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast. J Biol Chem 291:24261-24279
Mathew, Elizabeth; Dumont, Mark E (2015) A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs. Methods Enzymol 557:27-43
Zuber, Jeffrey; Danial, Shairy Azmy; Connelly, Sara M et al. (2015) Identification of destabilizing and stabilizing mutations of Ste2p, a G protein-coupled receptor in Saccharomyces cerevisiae. Biochemistry 54:1787-806
Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M et al. (2014) Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors. Biochim Biophys Acta 1838:15-33
Mathew, Elizabeth; Ding, Fa-Xiang; Naider, Fred et al. (2013) Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast. Protein Eng Des Sel 26:59-71
Gehret, Austin U; Connelly, Sara M; Dumont, Mark E (2012) Functional and physical interactions among Saccharomyces cerevisiae ýý-factor receptors. Eukaryot Cell 11:1276-88
Taslimi, Amir; Mathew, Elizabeth; Celic, Andjelka et al. (2012) Identifying functionally important conformational changes in proteins: activation of the yeast ýý-factor receptor Ste2p. J Mol Biol 418:367-78
Mathew, Elizabeth; Bajaj, Anshika; Connelly, Sara M et al. (2011) Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p. J Mol Biol 409:513-28