Exploration Of The Role Of Tyr57and Tyr185in Proton Transfer Process Of Bacteriorhodopsin

Bacteriorhodopsin (bR) is the main protein existing in halophilic bacteria membrane. It consists of248residues, and forms the structure composed of seven transmembrane a-helices. A retinal molecule covalently bound to the Lys216in the helix G, and the chromophore is selective absorption to light waves. As a light-driven proton pump, light is absorbed by retinal subsequently trigged changes in the conformation of the retinal. It is able to pump H+from cytoplasmic side of membrane to extrazellular space against electrochemical gradient. More studies show that aromatic amino acid residues in the membrane protein plays an important role on activation and function, but so far there is few reserches for them in bR.The advantages of the solid-state NMR is that the membrane proteins can be carried out in the natural lipid environment for their structure and functions, and able to be detected the activity state of the sample molecule, but also for directivity, dynamic, or biological molecules docked and other aspects of the study. While theoretically the method is not affected by molecular weightsize limit; and no special requirements to the form of the sample.In our project, aim to the role of aromatic residues Tyr57/Tyr185as proton co-receptor Asp212in regulating in the bR proton transfer process, we successly build the system for the expression and purify of mutants, and get four mutations concluding Y57F、Y57N、Y185I、Y185F. In addition, the protein samples are obtained through bringing up the wild-type R1M1stains and the mutant Y185F in culture with isotope-labeled [U-13C9,15N]-Tyr on the level of milligram. We use methods including UV spectrum scanning and dynamic spectroscopy for the reserch to the intermediate and photocycle of different Tyr mutants, and infer the possible effect of Y57/Y185to Asp212on the function. Through ssNMR and comparison experiments, taking preliminary assignment to Tyr residues in bR, discuss primarily the function of Y57/Y185, and get some meaningly conclusions, this laid a solid foundation for further research the function of the two residues.