Matthew J. Ranaghan

Protein-based artificial retinas

Artificial retinas based on the light transducing photoelectric protein bacteriorhodopsin exhibit differential responsivity, edge enhancement and motion detection. Under appropriate conditions, these artificial receptors mimic the differential responsivity characteristic of mammalian photoreceptor cells. The use of orientated bacteriorhodopsin to generate the photoelectrical signal provides rapid responsivity, high quantum efficiency and offers the potential of directly coupling the protein response to charge-sensitive semiconductor arrays. The ability to manipulate the properties of the protein via chemical and genetic methods enhances design flexibility.

meso-Arylporpholactones and their Reduction Products

The rational syntheses of meso-tetraaryl-3-oxo-2-oxaporphyrins 5, known as porpholactones, via MnO(4)(-)-mediated oxidations of the corresponding meso-tetraaryl-2,3-dihydroxychlorins (7) is detailed. Since chlorin 7 is prepared from the parent porphyrin 1, this amounts to a 2-step replacement of a pyrrole moiety in 1 by an oxazolone moiety. The stepwise reduction of the porpholactone 5 results in the formation of chlorin analogues, meso-tetraaryl-3-hydroxy-2-oxachlorin (11) and meso-tetraaryl-2-oxachlorins (12). The reactivity of 11 with respect to nucleophilic substitution by O-, N-, and S-nucleophiles is described. The profound photophysical consequences of the formal replacement of a pyrrole with an oxazolone (porphyrin-like chromophore) or (substituted) oxazole moiety (chlorin-like chromophore with, for the parent oxazolochlorin 12, red-shifted Q(x) band with enhanced oscillator strengths) are detailed and rationalized on the basis of SAC-CI and MNDO-PSDCI molecular orbital theory calculations. The single crystal X-ray structures of the porpholactones point at a minor steric interaction between the carbonyl oxygen and the flanking phenyl group. The essentially planar structures of all chromophores in all oxidation states prove that the observed optical properties originate from the intrinsic electronic properties of the chromophores and are not subject to conformational modulation.

Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

Over 4000 putative proteorhodopsins (PRs) have been identified throughout the oceans and seas of the Earth. The first of these eubacterial rhodopsins was discovered in 2000 and has expanded the family of microbial proton pumps to all three domains of life. With photophysical properties similar to those of bacteriorhodopsin, an archaeal proton pump, PRs are also generating interest for their potential use in various photonic applications. We perform here the first reconstitution of the minimal photoactive PR structure into nanoscale phospholipid bilayers (nanodiscs) to better understand how protein-protein and protein-lipid interactions influence the photophysical properties of PR. Spectral (steady-state and time-resolved UV-visible spectroscopy) and physical (size-exclusion chromatography and electron microscopy) characterization of these complexes confirms the preparation of a photoactive PR monomer within nanodiscs. Specifically, when embedded within a nanodisc, monomeric PR exhibits a titratable pK(a) (6.5-7.1) and photocycle lifetime (∼100-200 ms) that are comparable to the detergent-solubilized protein. These ndPRs also produce a photoactive blue-shifted absorbance, centered at 377 or 416 nm, that indicates that protein-protein interactions from a PR oligomer are required for a fast photocycle. Moreover, we demonstrate how these model membrane systems allow modulation of the PR photocycle by variation of the discoidal diameter (i.e., 10 or 12 nm), bilayer thickness (i.e., 23 or 26.5 Å), and degree of saturation of the lipid acyl chain. Nanodiscs also offer a highly stable environment of relevance to potential device applications.

Directed evolution of bacteriorhodopsin for applications in bioelectronics

In nature, biological systems gradually evolve through complex, algorithmic processes involving mutation and differential selection. Evolution has optimized biological macromolecules for a variety of functions to provide a comparative advantage. However, nature does not optimize molecules for use in human-made devices, as it would gain no survival advantage in such cooperation. Recent advancements in genetic engineering, most notably directed evolution, have allowed for the stepwise manipulation of the properties of living organisms, promoting the expansion of protein-based devices in nanotechnology. In this review, we highlight the use of directed evolution to optimize photoactive proteins, with an emphasis on bacteriorhodopsin (BR), for device applications. BR, a highly stable light-activated proton pump, has shown great promise in three-dimensional optical memories, real-time holographic processors and artificial retinas.

A second-site suppressor of a folding defect functions via interactions with a chaperone network to improve folding and assembly in vivo

Single amino acid substitutions in a protein can cause misfolding and aggregation to occur. Protein misfolding can be rescued by second‐site amino acid substitutions called suppressor substitutions (su), commonly through stabilizing the native state of the protein or by increasing the rate of folding. Here we report evidence that su substitutions that rescue bacteriophage P22 temperature‐sensitive‐folding (tsf) coat protein variants function in a novel way. The ability of tsf:su coat proteins to fold and assemble under a variety of cellular conditions was determined by monitoring levels of phage production. The tsf:su coat proteins were found to more effectively utilize P22 scaffolding protein, an assembly chaperone, as compared with their tsf parents. Phage‐infected cells were radioactively labelled to quantify the associations between coat protein variants and folding and assembly chaperones. Phage carrying the tsf:su coat proteins induced more GroEL and GroES, and increased formation of protein:chaperone complexes as compared with their tsf parents. We propose that the su substitutions result in coat proteins that are more assembly competent in vivo because of a chaperone‐driven kinetic partitioning between aggregation‐prone intermediates and the final assembled state. Through more proficient use of this chaperone network, the su substitutions exhibit a novel means of suppression of a folding defect.

Photochemical and Thermal Stability of Green and Blue Proteorhodopsins: Implications for Protein-Based Bioelectronic Devices

The photochemical and thermal stability of the detergent-solubilized blue- and green-absorbing proteorhodpsins, BPR and GPR, respectively, are investigated to determine the viability of these proteins for photonic device applications. Photochemical stability is studied by using pulsed laser excitation and differential UV-vis spectroscopy to assign the photocyclicity. GPR, with a cyclicity of 7 × 10(4) photocycles protein(-1), is 4-5 times more stable than BPR (9 × 10(3) photocycles protein(-1)), but is less stable than native bacteriorhodopsin (9 × 10(5) photocycles protein(-1)) or the 4-keto-bacteriorhodopsin analogue (1 × 10(5) photocycles protein(-1)). The thermal stabilities are assigned by using differential scanning calorimetry and thermal bleaching experiments. Both proteorhodopsins display excellent thermal stability, with melting temperatures above 85 °C, and remain photochemically stable up to 75 °C. The biological relevance of our results is also discussed. The lower cyclicity of BPR is found to be adequate for the long-term biological function of the host organism at ocean depths of 50 m or more.

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.

Optical applications of biomolecules

Abstract: From kinematics to molecular machines, biologically inspired technologies harness and enhance the intrinsic properties of naturally occurring materials and systems for applied technologies. Bacteriorhodopsin (BR) represents the most studied protein for photonic applications, and has found use in artificial retinas, associative and volumetric memories, optical limiters, photovoltaic cells and other devices. The native BR protein is rarely optimal for device applications, and genetic engineering plays an important role in the optimization process. In addition, new retinal proteins such as proteorhodopsin and channelrhodopsin-2 have been discovered which provide new options and opportunities.