Many proteins are built from structurally distinct subunits that communicate with each other by means of a conformational change in order to achieve overall function. The primary goal of this project is to create a new class of bi-functional, two-domain proteins that capture the properties of this conformationally-driven allosteric switch.
This aim will be accomplished by implementing the novel concept of mutually exclusive folding, in which the free energy stored in the native structure of one subunit is used to drive unfolding of another subunit within the same molecule. A fusion protein is created by inserting one protein into a surface loop of another. A topological constraint causes the two domains to engage in a thermodynamic tug-of-war, from which only one can emerge in its folded state at any given-time. They cannot simultaneously exist in their native states. This conformational equilibrium cooperative, reversible, and controllable by ligand binding serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The unique properties afforded by mutually exclusive folding will be additionally exploited to develop two new applications. The first is an Escherichia coil based approach for rapidly selecting ultra-stable protein variants in vivo. The mutually exclusive folding design, combined with the use of a cytotoxic enzyme for one of the protein domains, results in a selection method of unprecedented versatility and throughput. The second is a class of cytotoxic enzymes that kills specific cell types. By virtue of the mutually exclusive folding design, activity of the catalytic domain is turned on or off by binding of a ligand to an engineered regulatory domain. Ligand binding domains from any one of a large number of proteins can perform this function. This switching mechanism forms the basis for developing cytotoxic proteins that are activated by a wide variety of cell-specific effector molecules, and can thus target cancerous or virally infected cells for destruction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM069755-01A1
Application #
6823135
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
2004-07-01
Project End
2008-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
1
Fiscal Year
2004
Total Cost
$273,600
Indirect Cost
Name
Upstate Medical University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Ha, Jeung-Hoi; Karchin, Joshua M; Walker-Kopp, Nancy et al. (2015) Engineered Domain Swapping as an On/Off Switch for Protein Function. Chem Biol 22:1384-93
Zheng, Huimei; Bi, Jing; Krendel, Mira et al. (2014) Converting a binding protein into a biosensing conformational switch using protein fragment exchange. Biochemistry 53:5505-14
Ha, Jeung-Hoi; Shinsky, Stephen A; Loh, Stewart N (2013) Stepwise conversion of a binding protein to a fluorescent switch: application to Thermoanaerobacter tengcongensis ribose binding protein. Biochemistry 52:600-12
Ha, Jeung-Hoi; Loh, Stewart N (2012) Protein conformational switches: from nature to design. Chemistry 18:7984-99
Ha, Jeung-Hoi; Karchin, Joshua M; Walker-Kopp, Nancy et al. (2012) Engineering domain-swapped binding interfaces by mutually exclusive folding. J Mol Biol 416:495-502
Stratton, Margaret M; Loh, Stewart N (2011) Converting a protein into a switch for biosensing and functional regulation. Protein Sci 20:19-29
Stratton, Margaret M; McClendon, Sebastian; Eliezer, David et al. (2011) Structural characterization of two alternate conformations in a calbindin D?k-based molecular switch. Biochemistry 50:5583-9
Stratton, Margaret M; Loh, Stewart N (2010) On the mechanism of protein fold-switching by a molecular sensor. Proteins 78:3260-9
Stratton, Margaret M; Cutler, Thomas A; Ha, Jeung-Hoi et al. (2010) Probing local structural fluctuations in myoglobin by size-dependent thiol-disulfide exchange. Protein Sci 19:1587-94
Mitrea, Diana M; Parsons, Lee S; Loh, Stewart N (2010) Engineering an artificial zymogen by alternate frame protein folding. Proc Natl Acad Sci U S A 107:2824-9

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