Retroengineering Bacteriorhodopsins for Technological Applications

Vencatesan Renugopalakrishnan1-3 and Pingzuo Li3,4

1BioFold Inc., 1050 Crown Pointe Parkway, Suite 1700, Atlanta, GA 30338, U.S.A.,
2Children's Hospital, Harvard Medical School, Boston, MA 02115, U.S.A.,
3Florida International University, Miami, FL 33174, U.S.A., and
4Chinese Academy of Sciences, Shanghai 200233, P.R. China




The chromophoric group of bacteriorhodopsin consists of the retinylidene residue and an inner shell of interacting amino acids. Since the photocycle of br contains several thermal steps, the overall photocycle shows a dependence on temperature, pH. Metastable, blue shifted M state, has been the principal focus in most holographic applications of bacteriorhodopsin. It has been shown that the O intermediate converts itself into P and Q states. These two photointermediates assume significance when it comes to the design of bacteriorhodopsin mutants with long-lived intermediates. We have taken the view that the design of intermediates with high quantum yield can be better accomplished by a combination of theoretical analysis of the energy level diagram of wild-type bacteriorhodopsin and genetic engineering methods. One of the mutants, br192, that satisfies the criterion of high quantum efficiency for the branched photochemistry (O->P) and further the criterion of thermal robustness involves multiple mutations. We have used computer-aided rational design concept followed by experimental genetic manipulation of the respective cDNA's of bacteriorhodopsin to express the mutants E. coli and more recently in P. pastoris.