In the last ten years, green fluorescent protein (GFP) has changed from a nearly unknown protein to a frequently used tool in molecular biology, medicine and cell biology. The most common uses of GFP are as a fusion tag to visualize dynamic cellular events and to monitor protein localization, and as a report gene to monitor gene expression. GFP is commonly used in medicinal research. For example, GFP has been used as a marker for tumor cells, to illuminate tumor progression and allow for detection of metastases down to the single cell level.Computational methods will be used to investigate the following phenomena: * Yellow Fluorescent Proteins are GFP mutants that have lower fluorescent quantum yields than GFP's. The conformational freedom of the chromophore might be responsible for the lower quantum yields. * Mature GFP; i.e. fully fluorescent GFP, is most efficiently formed at temperatures well below 37xC. Computational methods will be used to search for mutants that mature more efficiently at higher temperatures and to establish the mechanism of their action. * GFP has an 11-sheet beta-barrel structure. Water and halide ions can enter the barrel. The pathways into the barrel can be found with activated molecular dynamics. The resultant information could be used to design more specific halide ion sensors. * DsRed is a red-emitting fluorescent protein from discosoma that is commercially available and has been studied in some detail. Two of the largest disadvantages of DsRed are its slow chromophore formation and the fact that DsRed is an obligate tetramer. These drawbacks could be overcome by computationally designing DsRed mutants that do not form multimeric structures, or by designing GFP mutants that can form chromophores with extended conjugation such as that found in DsRed. * The autocatalytic chromophore forming reaction of the purple GFP-like protein from Anemonia sulcata is significantly different to that observed in GFP. Computational methods will be used to investigate structural aspects of the chromophore formation in the purple GFP-Iike protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15GM059108-02
Application #
6503161
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Chin, Jean
Project Start
1999-04-01
Project End
2005-08-31
Budget Start
2002-09-01
Budget End
2005-08-31
Support Year
2
Fiscal Year
2002
Total Cost
$121,804
Indirect Cost
Name
Connecticut College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
New London
State
CT
Country
United States
Zip Code
06320
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
Ong, Wayne J-H; Alvarez, Samuel; Leroux, Ivan E et al. (2011) Function and structure of GFP-like proteins in the protein data bank. Mol Biosyst 7:984-92
Samma, Alex A; Johnson, Chelsea K; Song, Shuang et al. (2010) On the origin of fluorescence in bacteriophytochrome infrared fluorescent proteins. J Phys Chem B 114:15362-9
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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