Nicole L. Wagner

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.

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.

Effect of Molecular Symmetry on the Spectra and Dynamics of the Intramolecular Charge Transfer (ICT) State of Peridinin

The spectroscopic properties and dynamics of the excited states of two different synthetic analogues of peridinin were investigated as a function of solvent polarity using steady-state absorption, fluorescence, and ultrafast time-resolved optical spectroscopy. The analogues are denoted S-1- and S-2-peridinin and differ from naturally occurring peridinin in the location of the lactone ring and its associated carbonyl group, known to be obligatory for the observation of a solvent dependence of the lifetime of the S(1) state of carotenoids. Relative to peridinin, S-1- and S-2-peridinin have their lactone rings two and four carbons more toward the center of the π-electron system of conjugated carbon-carbon double bonds, respectively. The present experimental results show that as the polarity of the solvent increases, the steady-state spectra of the molecules broaden, and the lowest excited state lifetime of S-1-peridinin changes from ∼155 to ∼17 ps which is similar to the magnitude of the effect reported for peridinin. The solvent-induced change in the lowest excited state lifetime of S-2-peridinin is much smaller and changes only from ∼90 to ∼67 ps as the solvent polarity is increased. These results are interpreted in terms of an intramolecular charge transfer (ICT) state that is formed readily in peridinin and S-1-peridinin, but not in S-2-peridinin. Quantum mechanical computations reveal the critical factors required for the formation of the ICT state and the associated solvent-modulated effects on the spectra and dynamics of these molecules and other carbonyl-containing carotenoids and polyenes. The factors are the magnitude and orientation of the ground- and excited-state dipole moments which must be suitable to generate sufficient mixing of the lowest two excited singlet states.

Energetics and dynamics of the low-lying electronic states of constrained polyenes: implications for infinite polyenes.

Steady-state and ultrafast transient absorption spectra were obtained for a series of conformationally constrained, isomerically pure polyenes with 5-23 conjugated double bonds (N). These data and fluorescence spectra of the shorter polyenes reveal the N dependence of the energies of six (1)B(u)(+) and two (1)A(g)(-) excited states. The (1)B(u)(+) states converge to a common infinite polyene limit of 15,900 ± 100 cm(-1). The two excited (1)A(g)(-) states, however, exhibit a large (~9000 cm(-1)) energy difference in the infinite polyene limit, in contrast to the common value previously predicted by theory. EOM-CCSD ab initio and MNDO-PSDCI semiempirical MO theories account for the experimental transition energies and intensities. The complex, multistep dynamics of the 1(1)B(u)(+) → 2(1)A(g)(-) → 1(1)A(g)(-) excited state decay pathways as a function of N are compared with kinetic data from several natural and synthetic carotenoids. Distinctive transient absorption signals in the visible region, previously identified with S* states in carotenoids, also are observed for the longer polyenes. Analysis of the lifetimes of the 2(1)A(g)(-) states, using the energy gap law for nonradiative decay, reveals remarkable similarities in the N dependence of the 2(1)A(g)(-) decay kinetics of the carotenoid and polyene systems. These findings are important for understanding the mechanisms by which carotenoids carry out their roles as light-harvesting molecules and photoprotective agents in biological systems.

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.

Low-Temperature Trapping of Photointermediates of the Rhodopsin E181Q Mutant.

Three active-site components in rhodopsin play a key role in the stability and function of the protein: 1) the counter-ion residues which stabilize the protonated Schiff base, 2) water molecules, and 3) the hydrogen-bonding network. The ionizable residue Glu-181, which is involved in an extended hydrogen-bonding network with Ser-186, Tyr-268, Tyr-192, and key water molecules within the active site of rhodopsin, has been shown to be involved in a complex counter-ion switch mechanism with Glu-113 during the photobleaching sequence of the protein. Herein, we examine the photobleaching sequence of the E181Q rhodopsin mutant by using cryogenic UV-visible spectroscopy to further elucidate the role of Glu-181 during photoactivation of the protein. We find that lower temperatures are required to trap the early photostationary states of the E181Q mutant compared to native rhodopsin. Additionally, a Blue Shifted Intermediate (BSI, λmax = 498 nm, 100 K) is observed after the formation of E181Q Bathorhodopsin (Batho, λmax = 556 nm, 10 K) but prior to formation of E181Q Lumirhodopsin (Lumi, λmax = 506 nm, 220 K). A potential energy diagram of the observed photointermediates suggests the E181Q Batho intermediate has an enthalpy value 7.99 KJ/mol higher than E181Q BSI, whereas in rhodopsin, the BSI is 10.02 KJ/mol higher in enthalpy than Batho. Thus, the Batho to BSI transition is enthalpically driven in E181Q and entropically driven in native rhodopsin. We conclude that the substitution of Glu-181 with Gln-181 results in a significant perturbation of the hydrogen-bonding network within the active site of rhodopsin. In addition, the removal of a key electrostatic interaction between the chromophore and the protein destabilizes the protein in both the dark state and Batho intermediate conformations while having a stabilizing effect on the BSI conformation. The observed destabilization upon this substitution further supports that Glu-181 is negatively charged in the early intermediates of the photobleaching sequence of rhodopsin.

The Forbidden 11Bu– Excited Singlet State in Peridinin and Peridinin Analogues

Theoretical studies have predicted the presence of a forbidden 11Bu- state in proximity to the strongly allowed 11Bu+ excited state in polyenes and carotenoids. The 11Bu- state is invariably predicted to have a very low oscillator strength, which precludes direct optical spectroscopic assignment. We report here a direct UV-vis optical spectroscopic feature assigned to the 11Bu- state of S-2-peridinin, a synthetic analogue of the naturally occurring carotenoid, peridinin. The shift of the ground state dipole of S-2-peridinin compared to natural peridinin enhances the oscillator strength of absorption from the ground state to the 11Bu- state by 2 orders of magnitude relative to peridinin. It is postulated that this is due to a quadrupolar electrostatic field generated from the more central location of the lactone ring along the polyene chain in S-2-peridinin. MNDO-PSDCI and EOM-CCSD calculations provide a theoretical basis for this assignment and explain the unique properties of the 11Bu- state and why the transition from the ground state to this state has such a low oscillator strength in most other polyenes and carotenoids.

Pixel Characterization of a Protein-Based Retinal Implant Using a Microfabricated Sensor Array

Retinal degenerative diseases are characterized by the loss of photoreceptor cells within the retina and affect 30-50 million people worldwide. Despite the availability of treatments that slow the progression of degeneration, affected patients will go blind. Thus, there is a significant need for a prosthetic that is capable of restoring functional vision for these patients. The protein-based retinal implant offers a high-resolution option for replacing the function of diseased photoreceptor cells by interfacing with the underlying retinal tissue, stimulating the remaining neural network, and transmitting this signal to the brain. The retinal implant uses the photoactive protein, bacteriorhodopsin, to generate an ion gradient in the subretinal space that is capable of activating the remaining bipolar and ganglion cells within the retina. Bacteriorhodopsin can also be photochemically driven to an active (bR) or inactive (Q) state, and we aim to exploit this photochemistry to mediate the activity of pixels within the retinal implant. In this study, we made use of a novel retinomorphic foveated image sensor to characterize the formation of active and inactive pixels within a protein-based retinal implant, and have measured a significant difference between the output frequencies associated with the bR and Q states.

Combined optical and chemical asynchronous event pixel array

The paper presents the concept, layout and simulation results for a combined chemical and optical 92×92 pixel array designed as a 3.18×3.18mm2 ASIC in standard AMS 0.35μm CMOS technology. The optical pixels employ PN junctions as photodiodes, while the chemical pixels use ISFETs that are pH sensitive with the standard passivation layer of the process. Local light intensity and pH level are encoded using pulse frequency modulation (PFM) and conveyed off-chip using the address event representation (AER) protocol. The chip has been conceived specifically to characterize a light sensitive biomolecular thin film that is in turn intended as a retinal implant for the treatment of end-stage retinal degenerative diseases.