Kevin J. Wise

Optimization of bacteriorhodopsin for bioelectronic devices

Bacteriorhodopsin (BR) is the photoactive proton pump found in the purple membrane of the salt marsh archaeon Halobacterium salinarum. Evolution has optimized this protein for high photochemical efficiency, thermal stability and cyclicity, as the organism must be able to function in a hot, stagnant and resource-limited environment. Photonic materials generated via organic chemistry have yet to surpass the native protein in terms of quantum efficiency or cyclicity. However, the native protein still lacks the overall efficiency necessary for commercial viability and virtually all successful photonic devices using bacteriorhodopsin are based on chemical or genetic variants of the native protein. We show that genetic engineering can provide significant improvement in the device capabilities of proteins and, in the case of bacteriorhodopsin, a 700-fold improvement has been realized in volumetric data storage. We conclude that semi-random mutagenesis and directed evolution will play a prominent role in future efforts in bioelectronic optimization.

Volumetric optical memory based on bacteriorhodopsin

An architecture for a three-dimensional optical computer memory based on the photoactive protein bacteriorhodopsin (BR) is described, utilizing a branch-off of the BR photocycle to access a long-lived photointermediate capable of serving as an active element for memory storage. This intermediate (the Q-state) is accessed as a result of the sequential absorption of two photons, the first to initiate the BR photocycle, and the second to drive the protein into the branched photocycle from the O-state several milliseconds later. The stability of the Q-state arises predominantly from the fact that it is strongly blue-shifted with respect to other intermediates in the photocycle, making it invisible to the laser wavelengths used to write and read information in the memory. Both proof of principle and second-generation prototypes are currently being developed in the W.M. Keck Center for Molecular Electronics, in collaboration with Critical Link, LLC of Syracuse, NY. The article will focus on the BR-branched photocycle memory architecture, the remaining challenges to fabrication of a commercially viable device, and the ongoing efforts in prototype development, optimization and protein characterization at Syracuse University.

Optimization of protein-based volumetric optical memories and associative processors by using directed evolution

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.

Directed evolution of bacteriorhodopsin for device applications.

Publisher Summary This chapter discusses the use of directed evolution of bacteriorhodopsin (BR) for device applications. This protein is synthesized by the salt marsh archaeon Halobacterium salinarum when oxygen availability drops below a level sufficient to sustain respiration. The organism then switches spontaneously to photosynthetic energy production by generating a purple membrane containing BR. It is found that when BR absorbs light it pumps a proton, and the pH gradient is utilized to convert ADP to ATP. Site-directed mutants are constructed using the Stratagene Quik-Change mutagenesis system. Construction of BR variants for expression in T. thermophilus is achieved using a combination of mutagenesis methods. Inserting mutant bop fragments into the multiple cloning site of pMKE1 is the final step in the construction of a BR thermoselection vector. A combination of mutagenesis techniques are used to create randomly mutated regions in the bop gene. It is found that the thermal stability of BR is achieved by expressing randomly mutated proteins in an extremely thermophilic background.

Biomolecular Electronic Device Applications of Bacteriorhodopsin

Over the last thirty years, bacteriorhodopsin has become one of the most actively researched proteins in biochemistry and biophysics. Interest in this protein stems not only from its unique photochemistry as a light-driven proton pump, but also from its potential as an active component of biomolecular device applications. Architectures for devices that range in form and function from polymer film based holographic interferometers to three-dimensional optical memories have been proposed and built. Bacteriorhodopsin’s propensity for biomolecular optics and electronics is due to three main light-induced responses: (1) A light-induced photocycle that consists of a variety of photochemically-distinct intermediates characterized by shifted absorption maxima, (2) A branched photocycle that results in a permanent blue shifted intermediate, and (3) A complex time-dependent photoelectric response that varies in both sign and amplitude over several orders of magnitude. These properties will be discussed below, as relevant to the development of two specific bacteriorhodopsin applications: the branched photocycle three-dimensional optical memory and the holographic associative memory.

Photochromic Bacteriorhodopsin Mutant with High Holographic Efficiency and Enhanced Stability via a Putative Self-Repair Mechanism

The Q photoproduct of bacteriorhodopsin (BR) is the basis of several biophotonic technologies that employ BR as the photoactive element. Several blue BR (bBR) mutants, generated by using directed evolution, were investigated with respect to the photochemical formation of the Q state. We report here a new bBR mutant, D85E/D96Q, which is capable of efficiently converting the entire sample to and from the Q photoproduct. At pH 8.5, where Q formation is optimal, the Q photoproduct requires 65 kJ mol-1 of amber light irradiation (590 nm) for formation and 5 kJ mol-1 of blue light (450 nm) for reversion, respectively. The melting temperature of the resting state and Q photoproduct, measured via differential scanning calorimetry, is observed at 100 °C and 89 °C at pH 8.5 or 91 °C and 82 °C at pH 9.5, respectively. We hypothesize that the protein stability of D85E/D96Q compared to other blue mutants is associated with a rapid equilibrium between the blue form E85(H) and the purple form E85(−) of the protein, the latter providing enhanced structural stability. Additionally, the protein is shown to be stable and functional when suspended in an acrylamide matrix at alkaline pH. Real-time photoconversion to and from the Q state is also demonstrated with the immobilized protein. Finally, the holographic efficiency of an ideal thin film using the Q state of D85E/D96Q is calculated to be 16.7%, which is significantly better than that provided by native BR (6–8%) and presents the highest efficiency of any BR mutant to date.

Optimizing Photoactive Proteins for Optoelectronic Environments by Using Directed Evolution

Genetic engineering has recently emerged as a popular tool for tailoring biological macromolecules to function in nonnative environments. Most protein optimization efforts have advanced in large part as a result of significant advances in the methods and procedures of genetic engineering, most notably, directed evolution. Directed evolution mimics natural selection by combining techniques in genetic modification with differential selection. Most protein engineering research focuses on improving the thermal and chemical properties of enzymatic proteins for pharmaceutical applications. However, the recent emergence of nanobiotechnology has led researchers to broaden the scope of directed evolution. This chapter describes a strategy for tailoring the electronic and photochemical properties of proteins for performance in device applications. Among the many photoactive proteins found in nature, bacteriorhodopsin and its eubacterial counterpart, proteorhodopsin, are two leading candidates for protein-based device applications. The intrinsic stability, branched photochemistry, and photovoltaic properties of bacteriorhodopsin and proteorhodopsin make both proteins excellent candidates for three-dimensional volumetric memories, real-time holographic media, protein-based semiconductor devices, and artificial retinas.

Protein-based volumetric memories

This paper will explore the use of the protein, bacteriorhodopsin, as the photoactive recording medium in an optical three-dimensional memory. Although this protein has been used previously as the photoactive medium in a number of three-dimensional architectures (e.g., holographic and two- photon), a sequential one-photon volumetric architecture employing a photochemical branching reaction characteristic of the protein is currently showing the most promise. This unique branching reaction allows for long-term data storage by the protein, and rigorously excludes unwanted photochemistry. During the past two years, two prototypes have been constructed, and the preliminary results look promising. The use of chemical modification and genetic engineering of the protein has improved data reliability by roughly five-fold, but reliability remains an issue. Some of the key problems will be discussed. In addition, the use of gray-scale and polarization multiplexing to increase the storage capacity will be examined.