Green fluorescent protein (GFP) and firefly luciferase(luc) are commonly used tracer proteins. They have applications in many areas of molecular biology, genetics, and cell biology where they have been used as fusion tags to visualize dynamic cellular events and to monitor protein localization, and as reporter genes to monitor gene expression. Both GFP and luc are commonly used in medicine. For example, to visualize tumor metastasis and angiogenesis, to monitor the spread of herpes simplex virus, to get a better understanding of the role of beta cells in diabetes development, and to examine the misfolding of proteins in early onset dystonia. They have also been used in the fight against bioterrorism e.g. to detect anthrax. I propose using computational methods to: Examine the autocatalytic chromophore formation in GFP. What is the structure of the precyclized form of wild-type GFP? What is the role of Arg96? The answers to these questions will hopefully help in designing mutants that form the chromophore more efficiently. Computationally design fluorescent nidogen mutants. Nidogen has a very similar tertiary structure to GFP, but it does not form a chromophore. What is the smallest set of mutations that can lead to chromophore formation in nidogen? * The effect of the protein matrix on chromophore rotation. The GFP chromophore only fluoresces when GFP is in its native conformation this is because the protein matrix restricts chromophore rotation. I would like to use molecular dynamics simulations to examine the low energy conformations available to a freely rotating chromophore in all the GFP and GFP-like proteins in the protein database. This includes chromophoric non- fluorescent proteins. This work will help us understand the photochemistry of GFP, especially the light and dark states. Use homology and conformational searching methods to generate the active conformations of luc from the crystal structure of its inactive form and the known active conformations of some of the members in its protein superfamily. The results should lead to a better understanding of the synthetase and monoxygenase functions of luc.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
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Special Emphasis Panel (ZRG1-BCMB-Q (90))
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Preusch, Peter C
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Connecticut College
Schools of Arts and Sciences
New London
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Kohrt, Dawn; Crary, Jennifer; Zimmer, Marc et al. (2014) CDK6 binds and promotes the degradation of the EYA2 protein. Cell Cycle 13:62-71
Zimmer, Matthew H; Li, Binsen; Shahid, Ramza S et al. (2014) Structural Consequences of Chromophore Formation and Exploration of Conserved Lid Residues amongst Naturally Occurring Fluorescent Proteins. Chem Phys 429:5-11
Li, Binsen; Shahid, Ramza; Peshkepija, Paola et al. (2012) Water Diffusion In And Out Of The ýý-Barrel Of GFP and The Fast Maturing Fluorescent Protein, TurboGFP. Chem Phys 392:143-148
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Zimmer, Marc (2009) GFP: from jellyfish to the Nobel prize and beyond. Chem Soc Rev 38:2823-32
Megley, Colleen M; Dickson, Luisa A; Maddalo, Scott L et al. (2009) Photophysics and dihedral freedom of the chromophore in yellow, blue, and green fluorescent protein. J Phys Chem B 113:302-8
Lemay, Nathan P; Morgan, Alicia L; Archer, Elizabeth J et al. (2008) The Role of the Tight-Turn, Broken Hydrogen Bonding, Glu222 and Arg96 in the Post-translational Green Fluorescent Protein Chromophore Formation. Chem Phys 348:152-160
Maddalo, Scott L; Zimmer, Marc (2006) The role of the protein matrix in green fluorescent protein fluorescence. Photochem Photobiol 82:367-72
Zimmer, Marc (2002) Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 102:759-81

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