Effects Of Mutagenesis On Functions Of Bacteriorhodopsin And Corresponding Protein-based Functional Materials

The state of arts of bioscience and biotechnology has provided an increasing prosperity in the development of not only biology but also material science. The combination of biotechnology and material science could lead to new functional materials. This Ph. D thesis is focused upon two membrane proteins, bacteriorhodopsin (BR) and archaerhodopsin (AR4), both of which contain a retinal and are photoresponsive. After illumination, the retinal proteins go through a photocycle and transfer a proton from the cytoplasmic side to the extracellular side. Those photosensitive proteins exhibite amazing potential applications when serve as building blocks of some functional materials.During the work of this Ph.D thesis, we tried to carry out site-specific mutagenesis and construct a series of point mutants. The mutants were expressed in a bacterio-opsin deficient halobacterial strain (L33) by gene engineering. The analysis of novel mutants shed light into the underlying mechanism of the light-driven proton pump. The composite of those proteins and a synthetic polymer was prepared, which afforded a prototype material for information storage. A photoresponsive protein microarray was also fabricated. A new function of BR was explored.The novel results are summarized as follows:(1) Six novel mutants of BR including K41E and D102K were generated via gene engineering to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake. The order is reversed in another retinal protein AR4, which has different 41th and 102th residues. Our results rule out that possibility and confirm that the pKa of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pKa of Asp96. Compared to the wild-type BR, the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor.(2) Another new mutant of BR with the 96th aspartic acid replaced by valine (D96V) was obtained, and a composite film of D96V in a synthetic polymer matrix poly(vinyl alcohol) (PVA) was prepared. Although valine is very hydrophobic, this point mutant kept the basic biological activities, namely, photoelectric and photochromic responses. Nevertheless, the lifetime of M intermediate in the BR mutant was nearly two orders of magnitude longer than that of wild-type BR in neutral aqueous solution, which benefits its potential application as an information material. The M lifetime was further significantly prolonged after embedding D96V into PVA hydrogels. It was also found that D96V is very sensitive to water content in comparison with wild-type BR and another BR mutant.(3) A new function of purple membrane (PM), non-fouling was found, and a pixel-architecture film of retinal proteins with biological activity and contrast of cell adhesion was prepared by an approach combining chemical, physical and biological technologies. Mammalian cells such as murine preosteocytes MC3T3-E1 were seeded on the PM film, and significant resistance of the film to cell adhesion was found. The resistance lasted as long as two weeks. So, we afford a unique biomacromolecular material with both photoresponsibility and non-fouling property. Oriented multilayers of purple membrane composed of BR and lipids were further patterned on an array of gold electrode pixels. In order to improve stability and resolution, the gene engineering technique was employed to make a mutant of the protein BR by replacing the 36th amino acid residue from aspartic acid to cysteine with a thiol end group ready to react with gold; electric sedimentation was used to guarantee the high probability of formation of the Au-S bond and meanwhile to orient BR; further chemical crosslinking was introduced among layers of purple membranes to significantly enhance photoelectrical signals while keeping high stability. The non-bound BR region was eventually washed out by detergent, and the remaining BR pixels were detergent resistant due to chemical crosslinking among BR layers and covalent binding between the multilayer to the substrate. The protein array was confirmed to keep both photoelectrical activity and contrast of cell adhesion.