Abstract
The potential use of proteins in device applications has advanced in large part due to significant advances in the methods and procedures of protein engineering, most notably, directed evolution. Directed evolution has been used to tailor a broad range of enzymatic proteins for pharmaceutical and industrial applications. Thermal stability, chemical stability, and substrate specificity are among the most common phenotypes targeted for optimization. However, in vivo screening systems for photoactive proteins have been slow in development. A high-throughput screening system for the photokinetic optimization of photoactive proteins would promote the development of protein-based field-effect transistors, artificial retinas, spatial light modulators, photovoltaic fuel cells, three-dimensional volumetric memories, and optical holographic processors. This investigation seeks to optimize the photoactive protein bacteriorhodopsin (BR) for volumetric optical and holographic memories. Semi-random mutagenesis and in vitro screening were used to create and analyze nearly 800 mutants spanning the entire length of the bacterio-opsin (bop) gene. To fully realize the potential of BR in optoelectronic environments, future investigations will utilize global mutagenesis and in vivo screening systems. The architecture for a potential in vivo screening system is explored in this study. We demonstrate the ability to measure the formation and decay of the red-shifted O-state within in vivo colonies of Halobacterium salinarum, and discuss the implications of this screening method to directed evolution.
Similar content being viewed by others
References
Vsevolodov, N. N. (1998). Biomolecular Electronics. An Introductionvia Photosensitive Proteins. Birkhauser; Boston, 1998.
Arnold, F. and Moore, J. C. (1997), Adv. Biochem. Eng. 58, 1–14.
Rubingh, D. N. (1997), Curr. Op. Biotech. 8, 417–422.
Kuchner, O. and Arnold, F. (1997), Trends Biotech. 15, 523–530.
Miyazaki, K. and Arnold, F. H. (1999), J. Mol. Evol. 49, 716–720.
Olsen, M., Iverson, B., and Georgiou, G. (2000), Curr. Op. Biotech. 11, 331–337.
Arnold, F., Wintrode, P. L., Miyazaki, K., and Gershenson, A. (2001), Trends Biochem. Sci. 26, 100–106.
Dalby, P. A. (2003), Curr. Opin. Struct. Biol. 13, 500–505.
Kirk, O., Borchert, T. V., and Fuglsang, C. C. (2002), Curr. Op. Biotech. 13, 345–351.
Morawski, B., Quan, S., and Arnold, F. (2001), Biotechnol. Bioeng. 76, 99–107.
Sterner, R. and Liebl, W. (2001), Crit. Rev. Biochem. Mol. Bio. 36, 39–106.
Whaley, S. R., English, D. S., Hu, E. L., Barbara, P. F., and Belcher, A. M. (2000), Nature 405, 665–668.
Wise, K. J., Gillespie, N. B., Stuart, J. A., Krebs, M. P., and Birge, R. R. (2002), T. Biotechnol. 20, 387–394.
Seeman, N. C. and Belcher, A. M. (2002), Proc. Natl. Acad. Sci. USA 99(Suppl 2), 6451–6455.
Hillebrecht, J. R., Wise, K. J., Koscielecki, J. F., and Birge, R. R. (2004), Methods Enzymol. 388, 333–347.
Xu, J., Bhattacharya, P., and Varo, G. (2004), Biosens. Bioelectron. 19, 885–892.
Li, Q., Stuart, J. A., Birge, R. R., Xu, J., Stickrath, A. B., and Bhattacharya, P. (2004). Biosens. Bioelectron. 19, 869–874.
Xu, J., Stickrath, A. B., Bhattacharya, P., et al. (2003), Biophys. J. 85, 1128–1134.
Koek, W. D., Bhattacharya, N., Braat, J. J., Chan, V. S., and Westerweel, J. (2004), Opt. Lett. 29, 101–103.
Hampp, N. (2000), Appl. Microbiol. Biotechnol. 53, 633–639.
Chen, Z. and Birge, R. R. (1993), Trends Biotech. 11, 292–300.
Chen, Z., Govender, D., Gross, R., and Birge, R. (1995), BioSystems 35, 145–151.
Martin, C. H., Chen, Z. P., and Birge, R. R. (1997), in Proc. Pacific Symp. Biocomputing, R. B. Altman, R. K. Dunker, L. Hunter and T. E. Klein, eds., World Scientific, Maui; pp. 268–279.
Birge, R. R., Gillespie, N. B., Izaguirre, E. W., et al. (1999), J. Phys. Chem. B 103, 10,746–10,766.
Stuart, J. A., Tallent, J. R., Tan, E. H. L., and Birge, R. R. (1996), Proc. IEEE Nonvol. Mem. Tech. (INVMTC) 6, 45–51.
Oesterhelt, D. and Stoeckenius, W. (1971), Nature (London). New Biol. 233, 149–152.
Lanyi, J. K. (1999), Int. Rev. Cytolo. 187, 161–202.
Sato, H., Takeda, K., Tani, K., et al. (1999), Acta Cryst. D Biol. Cryst. 55, 1251–1256.
Birge, R. R. (1981), Ann. Rev. Biophys. Bioeng. 10, 315–354.
Ebrey, T. G. (1993), Light Energy Transduction in Bacteriorhodopsin, CRC Press, Boca Raton, FL.
Popp, A., Wolperdinger, M., Hampp, N., Bräuchle, C., and Oesterhelt, D. (1993), Biophys. J. 65, 1449–1459.
Paek, E. G. and Psaltis, D. (1987), Opt. Eng. 26, 428–433.
Birge, R. R., Fleitz, P. A., Gross, R. B., et al. (1990), Proc. IEEE EMBS 12, 1788–1789.
Gross, R. B., Izgi, K. C., and Birge, R. R. (1992), Proc. SPIE 1662, 186–196.
Birge, R. R., Parsons, B., Song, Q. W., and Tallent, J. R. (1997), Protein-Based Three-Dimensional Memories and Associative Processors, Blackweel Science Ltd., Oxford.
Birge, B., Fleitz, P., Gross, R., et al. Spatial light modulators and optical associative memories based on bacteriorhodopsin. in Materials Research Society, Boston, MA, 1990.
Georgescu, R., Bandara, G., and Sun, L. (2003), Meth. Mol. Biol. 231, 75–83.
Krebs, M. P., Hauss, T., Heyn, M. P., RajBhandary, U. L., and Khorana, H. G. (1991), Proc. Natl. Acad. Sci. USA 88, 859–863.
Peck, R. F., Dassarma, S., and Krebs, M. P. (2000), Mol. Microbiol. 35, 667–676.
Dyall-Smith, M. (2004), The Halohandbook: Protocols for Halobacterial Genetics, Melbourne.
Oesterhelt, D. and Stoeckenius, W. (1973), Proc. Natl. Acad. Sci. USA 70, 2853–2857.
Baliga, N. S., Kennedy, S. P., Ng, W. V., Hood, L., and Dassarma, S. (2001), Proc. Natl. Acad. Sci. USA. 98, 2521–2525.
Gillespie, N. B., Wise, K. J., Ren, L., et al. (2002), J. Phys. Chem. B 106, 13,352–13,361.
Hampp, N., Popp, A., Bräuchle, C., and Oesterhelt, D. (1992), J. Phys. Chem. 96, 4679–4685.
Zscherp, C., Schlesinger, R., and Heberle, J. (2001), Biochem. and Biophys. Res. Commun. 283, 57–63.
Balashov, S., Imasheva, E., Ebrey, T., Chen, N., Menick, D., and Crouch, R. (1997), Biochemistry 36, 8671–8676.
Brown, L. S., Sasaki, J., Kandori, H., Maeda, A., Needleman, R., and Lanyi, J. K. (1995), J. Biol. Chem. 270, 27,122–27,126.
Author information
Authors and Affiliations
Additional information
These authors contributed equally to this work.
Rights and permissions
About this article
Cite this article
Hillebrecht, J.R., Koscielecki, J.F., Wise, K.J. et al. Optimization of protein-based volumetric optical memories and associative processors by using directed evolution. Nanobiotechnol 1, 141–151 (2005). https://doi.org/10.1385/NBT:1:2:141
Issue Date:
DOI: https://doi.org/10.1385/NBT:1:2:141