Proton Pump Of Archaerhodopsin And Protein/Polymer Functional Materials

This Ph. D thesis concerns an interdisciplinary research among fields of polymer materials, life science, and information science. The main content of this thesis is to compare the proton pumping mechanisms of two membrane proteins, bacteriorhodopsin (BR) and archaerhodopsin 4 (AR4), to modify and optimize their properties by genetic mutation, to investigate the influences of amino acid and lipid environment on their proton pumping behaviors, and to prepare the functional polymer materials containing these active proteins in potential application as an information material.Membrane proteins are very important to the energy transition, information recognition and delivery, material transfer between the cell. In the last three decades, few single biological objects have attracted as much general interest as the purple protein, BR. This small retinal-containing protein has long promised to be the Rosetta stone for deciphering how ion pumps work, people also see its bright future in technical application. AR4 is a retinal-containing membrane protein isolated from a strain of Halobacteria from a Tibetan salt lake. It acts as a light-driven proton pump like BR. Compared with BR, AR4 is still a riddle and its function is to be investigated further.BR is the first photochromic biomolecule in a technical application, and the preparation of the recording media with polymer is a key technology in its potential application. Further, to make AR4 competitive as an information materials is an open question.Based on this, the mail creative work and results of the present thesis include:(1) The pH dependence of photocycle in AR4 was examined, the underlying proton pumping mechanism was investigated and a weak-coupling model was suggested. The temporal sequence of AR4 reversed to "normal" - proton release preceding proton uptake - when the pH is increased above 8.6. The weakenedcoupling between the protonation of Asp85 and deprotonation of proton release complex accounts for the lack of early proton release at neutral pH and the reversed time sequence of proton release and uptake in AR4 compared to that in BR. Nevertheless the proton release complex (PRC) in AR4 effectively facilitates deprotonation of primary proton acceptor and recovery of initial state at neutral pH. Compared to BR, AR4 is an alkaline-shifted proton pump.(2) Triton X-100 was found to alter the temporal sequence of the light-induced proton uptake and release of AR4, and it was revealed that the self-assembly state could influence the proton pumping behaviors of AR4. This order of proton release and uptake is reversed in the AR4 monomer compared to that in AR4 assembly. Further, removal of Triton X-100 by Biobeads resulted in the recovery of the temporal order of proton release and uptake for AR4. This finding implies some subtle protein-lipid interactions and self-assembly effect of protein in adjusting the proton pumping behavior of AR4.(3) The different proton pumping behaviors were further compared among transformed AR4, wild type (WT) AR4 and WT BR. In a cooperation work, AR4 and BR genes were cloned and expressed in the H. salinarium strain L33, which does not synthesize BR and carotenoid naturally, and we found that AR4 expressed in 133 (transf. AR4) shows different properties of photocycle and proton pump from both WT AR4 and BR. The difference between transf. AR4 and BR is caused by their different amino acid sequence, while the difference between transf. AR4 and WT AR4 is due to their different expression system and lipid environment (purple membrane vs. claret-colored membrane). Thus, we proposed that some key amino acid residues and lipid environment in AR4 co-determine its reversed temporal sequence of proton release and uptake.(4) Then, two new mutants of BR and AR4, BR-D96V and AR4-D212G were obtained and their basic photocycle and proton pump properties were characterized. WT BR, BR-D96N and WT AR4 were employed for comparison. Asp96 is one of the most important amino acids affecting the proton transport through the proton channel of BR. Although Val is very hydrophobic, such a point mutant doest not alterthe basic folding structure of BR, for the mutant functions as a proton pump and exhibits light-dark-adaptation behavior similar to BR-WT. The lifetime of the M intermediate in BR-D96V is more than one order of magnitude higher than observed for BR-WT at neutral pH, but on the same order of magnitude as BR-D96N. Increasing medium pH causes a significant prolongation of the M-lifetime in BR-D96V. This genetic modification improves the properties for potential information storage of BR. D212G mutant of AR4 restores its order of proton release and uptake, suggesting Asp212 is important to the proton pump of AR4.(5) Last, the composite films of recombinant proteins and polymers were prepared and investigated preliminarily. We optimized the properties of AR4 in technical application by genetic engineering, enhanced the interactions between polymer and protein, and prepared uniform materials in nano-scale for light information processing and storage. The absorption spectrum of carotenoid existing in the surrounding lipid of AR4 covers the absorption of the protein and weakens its photochromic property, hindering its application in light information field. By the method of genetic engineering, carotenoid is removed and the photochromic property of AR4 is improved. Further genetic mutation optimizes its function. Composite films were made by embedding genetic engineered AR4 into polymer for device application. Simple pattern recording is also realized here, making AR4 a potential biomaterial for information storage. In materials preparation, thermal sensitive Pluronic etc. were employed to prepare BR containing composite film or hydrogel.So this thesis is based on the interdisciplinary research related to polymer, biological and information sciences. The picture of the proton pump for AR4 was portrayed, which is helpful for understanding the mechanism of ion pumps more extensively and enrich the knowledge of mankind; on the other hand, our own intellectual properties were obtained in the aspects of both new materials and new technique for materials preparation.