Jason R. Hillebrecht

Protein Flexibility and Conformational State: A Comparison of Collective Vibrational Modes of Wild-Type and D96N Bacteriorhodopsin

Far infrared (FIR) spectral measurements of wild-type (WT) and D96N mutant bacteriorhodopsin thin films have been carried out using terahertz time domain spectroscopy as a function of hydration, temperature, and conformational state. The results are compared to calculated spectra generated via normal mode analyses using CHARMM. We find that the FIR absorbance is slowly increasing with frequency and without strong narrow features over the range of 2-60 cm(-1) and up to a resolution of 0.17 cm(-1). The broad absorption shifts in frequency with decreasing temperature as expected with a strongly anharmonic potential and in agreement with neutron inelastic scattering results. Decreasing hydration shifts the absorption to higher frequencies, possibly resulting from decreased coupling mediated by the interior water molecules. Ground-state FIR absorbances have nearly identical frequency dependence, with the mutant having less optical density than the WT. In the M state, the FIR absorbance of the WT increases whereas there is no change for D96N. These results represent the first measurement of FIR absorbance change as a function of conformational state.

Direct Measurement of the Photoelectric Response Time of Bacteriorhodopsin via Electro-Optic Sampling

The photovoltaic signal associated with the primary photochemical event in an oriented bacteriorhodopsin film is measured by directly probing the electric field in the bacteriorhodopsin film using an ultrafast electro-optic sampling technique. The inherent response time is limited only by the laser pulse width of 500 fs, and permits a measurement of the photovoltage with a bandwidth of better than 350 GHz. All previous published studies have been carried out with bandwidths of 50 GHz or lower. We observe a charge buildup with an exponential formation time of 1.68 +/- 0.05 ps and an initial decay time of 31.7 ps. Deconvolution with a 500-fs Gaussian excitation pulse reduces the exponential formation time to 1.61 +/- 0.04 ps. The photovoltaic signal continues to rise for 4.5 ps after excitation, and the voltage profile corresponds well with the population dynamics of the K state. The origin of the fast photovoltage is assigned to the partial isomerization of the chromophore and the coupled motion of the Arg-82 residue during the primary event.

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.

Humidity-dependent open-circuit photovoltage from a bacteriorhodopsin/indium tin oxide bioelectronic heterostructure

Non-contact electrostatic force microscopy techniques were used to study the open-circuit photovoltage of purple membrane multilayers electrodeposited on indium tin oxide. A humidity-dependent photovoltage response under illumination by a 635 nm photodiode was observed. Peak photovoltages in excess of 2 V at ~36 W m−2 were obtained for ~15% relative humidity.

Soft lithography based micron-scale electrophoretic patterning of purple membrane

Soft lithography and thermal evaporation of gold were used to create microfluidic channels with metal only on the bottom surface of each channel, in a process similar to nanotransfer printing (nTP). Micron-scale patterns of the bioelectronic material purple membrane (PM) were fabricated by combining standard soft lithography with electrophoretic deposition. Each metallized microfluidic channel is converted into a micron-scale electrophoresis cell by filling with a PM solution and applying a small voltage. Any number of patterns can be created for a variety of bioelectronic device applications. While this study focuses exclusively on the patterning of PM, micro-electrophoretic sedimentation is applicable to a wide range of biological and organic macromolecules.

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.

Terahertz measurements of the Photoactive Protein Bacteriorhodopsin mutant D96N: M and P states

We use terahertz (THz) spectroscopy as a biomaterials characterization tool. Previously we have shown a strong contrast between the THz dielectric response for wild type (WT) and D96N mutant of bacteriorhodopsin. In those studies we observed a large increase in the THz absorbance of WT with excitation to thermally captured photo-intermediates whereas no such increase in absorbance was observed for the mutant D96N. These results suggest that the THz response is sensitive to structural changes and relative flexibility of biomolecules. However the photo-intermediate populations of the WT and D96N samples were not equivalent in those measurements. While the WT samples had relaxed (bR), M and P state intermediates present, the D96N samples had only bR and M states. Here we present terahertz absorbance measurements of D96N as a function of M and P state populations at room temperature. The THz response is constant for intermediate states populations up to 23% M state and up to 30% P state. These results verify that there is a fundamental difference in the conformational dynamics as measured by THz dielectric response for a single residue mutation.

Ultrafast spectroscopy of photoactive biomolecules

Terahertz (THz) absorbance measurements as a function of confirmation for bacteriorhodopsin has been performed in the steady state demonstrating the dependence on protein conformation and mutation. We introduce a new technique for performing low time resolution visible pump/THz probe measurements relevant to biomolecular systems. We will discuss the experimental protocol required for these measurements and how the THz absorbance can be used as a characterization tool for technologically important biomolecules.