Examination of the Hydrophobicity of the LOV (Light-Oxygen-Voltage) Protein Active Site, Using Computational Chemistry and Raman Spectroscopy

LOV (Light-Oxygen-Voltage) domains are common blue light sensing proteins, found in plants, fungi and both kingdoms of prokarya. LOV proteins are also commonly used in engineered proteins, usually to impart blue light sensitivity. Despite their ubiquity and utility, the mechanism by which they turn light into a biochemical signal is contested. The initial photophysics, and the allosteric protein effects are well documented; but the key chemical step in between these two processes is unknown. In this step, a triplet state on the flavin chromophore creates a chemical bond to a nearby cysteine. This thesis describes the use of computational chemistry and Raman spectroscopy to explore the role of water in the LOV photocycle. Both molecular dynamics, and quantum chemistry was applied to this effort. GROMACS was the MD engine used, and the CHARMM27 force field was employed. The quantum chemistry program Orca was chosen for the density functional theory described in this document. Both of these computational methods show that water is stable in the LOV active site. Raman vibrational spectroscopy of free cysteine as a model compound show that the catalytic Cys inside the LOV active site need not be protonated for LOV photochemistry to occur.