The goal of this project is to develop mechanisms by which protein folding can be coupled to unfolding, in order to introduce allosteric control into proteins and enzymes. Two techniques are introduced for this purpose: mutually exclusive folding (MEF) and alternate frame folding (AFF). MEF uses the free energy stored in the folded structure of one domain to unfold another domain within the same molecule. The two domains cannot exist simultaneously in their native states;hence the molecule interconverts between two functional forms. By matching a receptor domain with an enzymatic or functional domain, one can create new molecules in which ligand binding causes a desired biological function. AFF is a novel mechanism for coupling binding to conformational change in a simple, predictable, and well-defined manner. It involves permutation of the amino acid sequence and partial sequence duplication. The resulting protein switches between native structures that are structurally similar, but possess different topologies. The driving force is provided by the universal linkage between ligand binding and protein folding. The conformational change is harnessed to a biological function or to an output signal. AFF can be implemented within a single protein or can be combined with MEF in a two- domain fusion to establish allosteric regulation.
The aims will generate biosensors for calcium and sugars, protein scaffolds for development of versatile (""""""""universal"""""""") sensors, a potent anti-HIV toxin, and a method for introducing conditional functionality to a protein of choice. These molecules will establish a framework for designing a wide variety of proteins with novel molecular recognition and therapeutic properties.

Public Health Relevance

This project develops new mechanisms for introducing allosteric control into proteins. This technology is used to create proteins of medical significance (anti-HIV therapeutics) and biological interest (biosensors for metals, sugars, and other small molecules).

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-B (03))
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Wehrle, Janna P
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Upstate Medical University
Schools of Medicine
United States
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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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