Temperature Dependent Protein-Chromophore Hydrogen Bond Dynamics in the Far-Red Fluorescent Proteins by using Molecular Dynamics Simulation and Quantum CalculationTemperature Dependent Protein-Chromophore Hydrogen Bond Dynamics in the Far-Red Fluorescent Proteins by using Molecular Dynamics Simulation and Quantum Calculation
Doctor of Philosophy (PhD)
First Advisor's Name
Dr. Xuewen Wang
First Advisor's Committee Title
Committee chair: Dr. Xuewen Wang
Second Advisor's Name
Dr. Prem Chapagain
Second Advisor's Committee Title
Co-committee chair: Dr. Prem Chapagain
Third Advisor's Committee Title
Committee member: Dr. Jin He
Fourth Advisor's Committee Title
Committee member: Dr. Watson J. Lees
BioPhysics, Far-Red Fluorescent Proteins, Stokes shift
Date of Defense
Fluorescent proteins are valuable tools as biochemical markers in molecular and cell biology research for studying cellular processes. Red Fluorescent Proteins (RFPs) are highly desirable for in vivo applications in living cell imaging because they absorb and emit light in the red region of the spectrum where cellular autofluorescence. Naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. For their use as biochemical markers, several monomeric variants of RFP have been developed which include mCherry, dsRed, and mStrawberry. Far red-emitting FPs with large Stokes shift are especially valuables for in vivo applications due to the advantage of deep tissue penetration, longer imaging times, and low cellular autofluorescence. Examples of far-red emitting FPs include mPlum, mKate, TagRFP675, and more recently developed versions include mNeptune1, mNeptune2.5, and mCardinal2. Low-temperature experiments on mneptune1, mNeptune2.5, and mcardinal2 show a reduced Stokes shift compared to room temperature. To characterize their Stokes shift behavior at different temperatures, I used a combination of molecular dynamics (MD) simulations and quantum mechanical calculations and investigated the dynamics of the hydrogen bonds formed due to protein-chromophore interactions at different temperatures for these FPs. Since the switching between direct and water-mediated hydrogen bonds has been shown to correlate with the Stokes shift in a related protein mPlum, I investigated the agile switching of the chromophore-PHE62 hydrogen bond between the direct and water-mediated bonding in these FPs at various temperatures. MD simulations show that while all three variants show the ability to switch the water-mediated vs. direct hydrogen bonding at 300K, mCardinal2 shows better hydrogen bond flexibility. My results provide insights into the role of thermal fluctuations on the solvation and hydrogen bonding in the chromophore environment in the FP variants. The structures of the extended chromophore obtained from MD simulations were used to calculate the excitation and emission energy/wavelength using quantum mechanical calculation. These calculations were performed separately for the direct vs. water-mediated structures extracted from the simulation trajectories. My results show that the increased flexibility of the chromophore environment at higher temperatures along with its ability to reorganize after excitation is related to the larger Stokes shift.
Dhakal, Chandra Prasad, "Temperature Dependent Protein-Chromophore Hydrogen Bond Dynamics in the Far-Red Fluorescent Proteins by using Molecular Dynamics Simulation and Quantum CalculationTemperature Dependent Protein-Chromophore Hydrogen Bond Dynamics in the Far-Red Fluorescent Proteins by using Molecular Dynamics Simulation and Quantum Calculation" (2021). FIU Electronic Theses and Dissertations. 4837.
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