Colorado PROFILES, The Colorado Clinical and Translational Sciences Institute (CCTSI)
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ENGINEERING A YEAST SENSOR OF LIGAND-BINDING


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Collapse abstract
Small ligands play important roles in protein regulation, signal transduction, and other fundamental processes in the cell, and are the basis for pharmaceutical approaches to treatment of human disease. Simple and efficient methods for recognizing binding of small ligands, therefore, are in great demand, and would have applications in a number of areas, from high-throughput screening in drug discovery to structural studies of protein-ligand interactions. This proposal outlines plans for generating a yeast temperature-sensitive enzyme whose activity is regulated by ligand binding. This will be a hybrid enzyme containing a catalytic domain reporter and an inserted or tethered ligand-binding domain. The design is based on the premise that ligand binding to a domain may stabilize it or induce an allosteric change that may be detected by a catalytic domain if it is closely coupled. To identify proteins with coupled binding and catalytic domains, engineered constructs will be screened in yeast for forms that show altered reporter activity in the presence of ligand. Subtle changes in catalytic activity or stability can be detected in yeast by using a temperature-sensitive protein as a reporter. As an initial test of the feasibility of such a screen, temperature-sensitive dihydrofolate reductase (DHFR) will be coupled to the FK506 binding protein, FKBP12, and screened in yeast cells in the presence and absence of FK506. If this strategy is successful, ligand binding can be easily observed as a change in temperature sensitivity of the yeast cells. The hybrid protein will ideally have a modular design, such that other binding domains can be substituted to recognize other ligands.
Collapse sponsor award id
F32GM020532

Collapse Time 
Collapse start date
2000-09-01
Collapse end date
1900-01-01

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