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. (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
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
Cutler, Thomas A; Mills, Brandon M; Lubin, David J et al. (2009) Effect of interdomain linker length on an antagonistic folding-unfolding equilibrium between two protein domains. J Mol Biol 386:854-68
Butler, James S; Mitrea, Diana M; Mitrousis, Gregory et al. (2009) Structural and thermodynamic analysis of a conformationally strained circular permutant of barnase. Biochemistry 48:3497-507
Stratton, Margaret M; Mitrea, Diana M; Loh, Stewart N (2008) A Ca2+-sensing molecular switch based on alternate frame protein folding. ACS Chem Biol 3:723-32
Cutler, Thomas A; Loh, Stewart N (2007) Thermodynamic analysis of an antagonistic folding-unfolding equilibrium between two protein domains. J Mol Biol 371:308-16
Ha, Jeung-Hoi; Butler, James S; Mitrea, Diana M et al. (2006) Modular enzyme design: regulation by mutually exclusive protein folding. J Mol Biol 357:1058-62