A vibrational spectroscopy study of two proteorhodopsin variants

Proteorhodopsins (PRs) are a large family of membrane proteins that function as light-driven proton pumps which result in cellular energy storage and/or control of cellular processes such as phototaxis. Over 5000 proteorhodopsin variants have been found in bacteria across the world's oceans making it a significant source of solar energy absorption. The majority of PRs can be divided into two major classes based on their photochemical properties: green- and blue-absorbing (GPR and BPR, respectively). My thesis is directed in part towards understanding the molecular causes for the proteorhodopsin differentiation, and how those changes are used by the native organism. The primary technique used was vibrational spectroscopy by IR and Raman spectroscopy to determine the role of specific components of proteorhodopsins including the retinal chromophore, amino acid residues, backbone structure and internal water molecules.;I have identified several crucial differences between BPR and GPR. (1) I have found that the identity of residue 105 is the sole determinant of interactions between the protein and the retinal chromophore in the unphotolyzed dark state. In BPR a polar glutamine directly interacts with the Schiff base (SB) and C13 retinylidene methyl group causing a color shift. (2) PRs have a single highly conserved histidine located near the protein's photoactive site which was revealed to participate in the proton translocation mechanism of PR. In GPR the histidine residue is positively charged in the unphotolyzed state and undergoes a deprotonation during the photocycle. (3) Glutamic acid 142 is neutral in GPR, but is deprotonated and can act as an alternate proton acceptor in BPR only. We measured the pKa in BPR to be ∼7.7 which is close to the environmental pH of ∼8.0. These protein responses may be part of a light activated pH sense for the native marine bacteria.;With these data taken together I have proposed a model for PR function in BPR. Upon isomerization, the protonated Schiff base donates a proton either to Asp97 or G1u142 depending on the environmental pH. The proton is then relayed to Glu214 where it is ejected into the medium. In GPR the proton chain includes both Asp97 and His75.