The goal of this research is to improve the fluorescent properties of an important class of fluorescent proteins. Fluorescent proteins are extremely valuable biochemical markers in molecular and cell biology that allow monitoring of cellular biology on a molecular level. Fluorescent proteins are used to elucidate the biochemical pathways of healthy cells, and uncover the molecular problems that cause diseases. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. Naturally occurring RFPs are polymeric, which makes them unsuitable for use in tagging purposes. Structural modifications to these RFPs have produced monomeric RFP variants, but at present their usefulness is limited because they are not photostable. We hypothesize that due to the compromised structural integrity of monomeric RFPs, the chromophore is not well protected from attack by molecular oxygen. Computational investigations of structure-function relationships will allow the design of protein- chromophore monomeric RFP variants that are impermeable to oxygen for extended periods of time. These computationally designed monomeric RFP variants will assist experimentalists in preparing RFPs with chromophores that are better protected and with substantially improved photostability. This work will also develop tools for future computational design of monomeric RFPs with protein-chromophore combinations with improved brightness. The proposed research has three specific Aims: (1)Parameterization of force fields for various chromophores to use in computational studies (2)Investigation of the structural integrity and fluctuations of the proteins of monomeric variants of RFPs to identify key regions where oxygen can enter and attack the chromophore. (3)Identify protein regions with specific amino acids that can temporarily host oxygen and release it at the appropriate time to actively assist the debilitating transport of oxygen to the interior. Identifying these regions will permit a systematic computational-experimental strategy of amino acid substitution to create more protective proteins, and to modify the protein-chromophore interactions to enhance the fluorescent properties of the system.

Public Health Relevance

The goal of this research is to improve the usefulness of an important class of molecules called red fluorescent proteins. Fluorescent proteins are extremely valuable biochemical markers that allow the monitoring of cellular biology on a molecular level. Fluorescent proteins are used to elucidate the biochemical pathways of healthy cells, and uncover the molecular problems that cause diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Continuance Award (SC3)
Project #
5SC3GM096903-02
Application #
8306851
Study Section
Special Emphasis Panel (ZGM1-MBRS-X (CH))
Program Officer
Krasnewich, Donna M
Project Start
2011-07-25
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
2
Fiscal Year
2012
Total Cost
$108,750
Indirect Cost
$33,750
Name
Florida International University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
071298814
City
Miami
State
FL
Country
United States
Zip Code
33199
Dean, Kevin M; Lubbeck, Jennifer L; Davis, Lloyd M et al. (2015) Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching. Integr Biol (Camb) 7:263-73
Konold, Patrick; Regmi, Chola K; Chapagain, Prem P et al. (2014) Hydrogen bond flexibility correlates with Stokes shift in mPlum variants. J Phys Chem B 118:2940-8
Regmi, Chola K; Bhandari, Yuba R; Gerstman, Bernard S et al. (2013) Exploring the diffusion of molecular oxygen in the red fluorescent protein mCherry using explicit oxygen molecular dynamics simulations. J Phys Chem B 117:2247-53
Chapagain, Prem P; Regmi, Chola K; Castillo, William (2011) Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry. J Chem Phys 135:235101