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Compared with batch and vapor diffusion methods, counter diffusion can generate larger and higher‐quality protein crystals yielding improved diffraction data and higher‐resolution structures. Typically, counter‐diffusion experiments are conducted in elongated chambers, such as glass capillaries, and the crystals are either directly measured in the capillary or extracted and mounted at the X‐ray beamline. Despite the advantages of counter‐diffusion protein crystallization, there are few fixed‐target devices that utilize counter diffusion for crystallization. In this article, different designs of user‐friendly counter‐diffusion chambers are presented which can be used to grow large protein crystals in a 2D polymer microfluidic fixed‐target chip. Methods for rapid chip fabrication using commercially available thin‐film materials such as Mylar, propylene and Kapton are also detailed. Rules of thumb are provided to tune the nucleation and crystal growth to meet users' needs while minimizing sample consumption. These designs provide a reliable approach to forming large crystals and maintaining their hydration for weeks and even months. This allows ample time to grow, select and preserve the best crystal batches before X‐ray beam time. Importantly, the fixed‐target microfluidic chip has a low background scatter and can be directly used at beamlines without any crystal handling, enabling crystal quality to be preserved. The approach is demonstrated with serial diffraction of photoactive yellow protein, yielding 1.32 Å resolution at room temperature. Fabrication of this standard microfluidic chip with commercially available thin films greatly simplifies fabrication and provides enhanced stability under vacuum. These advances will further broaden microfluidic fixed‐target utilization by crystallographers. [ABSTRACT FROM AUTHOR]

Another way to access interfacial dynamics is to study vibrational transitions localized at an interface as reported by Lee I et al. i ,[11] who use the ester carbonyl of model lipids in bilayers in conjunction with MD simulations to characterize hydration structural dynamics. Yang I et al. i [22] have investigated an energetic material, cyclotetramethylene tetranitramine (HMX), using ultrafast transient absorption and 2D-IR spectroscopy of the nitro stretch as a probe, revealing structural dynamics as well as vibrational energy transfer. Solowan I et al. i [4] study azulene using action-based 2D spectroscopy in both the liquid and gas phases, finding differences in the energetics and the rates of conical intersection (CI) and internal conversion in the two conditions. The amide I mode of peptides and proteins offers a powerful means of accessing protein structural dynamics, and the contribution from Tan I et al. i [12] shows how the vibrational anharmonicity is a sensitive probe of protein-water coupling. [Extracted from the article]

Protein structural dynamics can span many orders of magnitude in time. Photoactive yellow protein's (PYP) reversible photocycle encompasses picosecond isomerization of the light-absorbing chromophore as well as large scale protein backbone motions occurring on a millisecond timescale. Femtosecond-to-millisecond time-resolved mid-infrared spectroscopy is employed here to uncover structural details of photocycle intermediates up to chromophore protonation and the first structural changes leading to the formation of the partially unfolded signaling state pB. The data show that a commonly thought stable transient photocycle intermediate is actually formed after a sequence of several smaller structural changes. We provide residue-specific spectroscopic evidence that protonation of the chromophore on a few hundreds of microseconds timescale is delayed with respect to deprotonation of the nearby E46 residue. That implies that the direct proton donor is not E46 but most likely a water molecule. Such details may assist the ongoing photocycle and protein folding simulation efforts on the complex and wide time-spanning photocycle of the model system PYP. [ABSTRACT FROM AUTHOR]

This article offers insights into PAS domains, which are sensor domains found in various signaling proteins and play a crucial role in signal transduction pathways. It discusses the origins and evolution of PAS domains across different organisms, highlighting their potential as drug targets for various diseases, including cancer.

We successfully created a composite photonic structure out of porous silicon (PSi) microcavities doped by the photochromic protein, photoactive yellow protein (PYP). Massive incorporation of the protein molecules into the pores was substantiated by a 30 nm shift of the resonance dip upon functionalization, and light-induced reflectance changes of the device due to the protein photocycle were recorded. Model calculations for the photonic properties of the device were consistent with earlier results on the nonlinear optical properties of the protein, whose degree of incorporation into the PSi structure was also estimated. The successful proof-of-concept results are discussed in light of possible practical applications in the future. [ABSTRACT FROM AUTHOR]

Chiral and achiral vibrational sum-frequency generation (VSFG) spectroscopy was performed in the 1400–1700 and 2800–3800 cm−1 range to study the interfacial structure of photoactive yellow protein (PYP) adsorbed on polyethyleneimine (PEI) and poly-l-glutamic acid (PGA) surfaces. Nanometer-thick polyelectrolyte layers served as the substrate for PYP adsorption, with 6.5-pair layers providing the most homogeneous surfaces. When the topmost material was PGA, it acquired a random coil structure with a small number of β2-fibrils. Upon adsorption on oppositely charged surfaces, PYP yielded similar achiral spectra. However, the VSFG signal intensity increased for PGA surfaces with a concomitant redshift of the chiral Cα-H and N–H stretching bands, suggesting increased adsorption for PGA compared to PEI. At low wavenumbers, both the backbone and the side chains of PYP induced drastic changes to all measured chiral and achiral VSFG spectra. Decreasing ambient humidity led to the loss of tertiary structure with a re-orientation of α-helixes, evidenced by a strongly blue-shifted chiral amide I band of the β-sheet structure with a shoulder at 1654 cm−1. Our observations indicate that chiral VSFG spectroscopy is not only capable of determining the main type of secondary structure of PYP, i.e., β-scaffold, but is also sensitive to tertiary protein structure. [ABSTRACT FROM AUTHOR]

A fundamental problem in biological sciences is understanding how macromolecular machines work and how the structural changes of a molecule are connected to its function. Time-resolved techniques are vital in this regard and essential for understanding the structural dynamics of biomolecules. Timeresolved small- and wide-angle X-ray solution scattering has the capability to provide a multitude of information about the kinetics and global structural changes of molecules under their physiological conditions. However, standard protocols for such time-resolved measurements often require significant amounts of sample, which frequently render time-resolved measurements impossible. A cytometry-type sheath co-flow cell, developed at the BioCARS 14-ID beamline at the Advanced Photon Source, USA, allows time-resolved pump-probe X-ray solution scattering measurements to be conducted with sample consumption reduced by more than ten times compared with standard sample cells and protocols. The comparative capabilities of the standard and coflow experimental setups were demonstrated by studying time-resolved signals in photoactive yellow protein. [ABSTRACT FROM AUTHOR]

We report an extension of a hybrid polarizable embedding method incorporating solvent effects in the calculations of two-photon absorption (2PA) cross sections. We employ the equation-of-motion coupled-cluster singles and doubles method for excitation energies (EOM-EE-CCSD) for the quantum region and the effective fragment potential (EFP) method for the classical region. We also introduce a rigorous metric based on 2PA transition densities for assessing the domain of applicability of QM/MM (quantum mechanics/molecular mechanics) schemes for calculating 2PA cross sections. We investigate the impact of the environment on the 2PA cross sections of low-lying transitions in microhydrated clusters of para-nitroaniline, thymine, and the deprotonated anionic chromophore of photoactive yellow protein (PYPb). We assess the performance of EOM-EE-CCSD/EFP by comparing the 2PA cross sections against full QM calculations as well as against the non-polarizable QM/MM electrostatic embedding approach. We demonstrate that the performance of QM/EFP improves when few explicit solvent molecules are included in the QM subsystem. We correlate the errors in the 2PA cross sections with the errors in the key electronic properties—identified by the analysis of 2PA natural transition orbitals and 2PA transition densities—such as excitation energies, transition moments, and dipole-moment differences between the initial and final states. Finally, using aqueous PYPb, we investigate the convergence of 2PA cross sections to bulk values. [ABSTRACT FROM AUTHOR]

Many physical phenomena must be accounted for to accurately model solution-phase optical spectral line shapes, from the sampling of chromophore-solvent configurations to the electronic-vibrational transitions leading to vibronic fine structure. Here we thoroughly explore the role of nuclear quantum effects, direct and indirect solvent effects, and vibronic effects in the computation of the optical spectrum of the aqueously solvated anionic chromophores of green fluorescent protein and photoactive yellow protein. By analyzing the chromophore and solvent configurations, the distributions of vertical excitation energies, the absorption spectra computed within the ensemble approach, and the absorption spectra computed within the ensemble plus zero-temperature Franck-Condon approach, we show how solvent, nuclear quantum effects, and vibronic transitions alter the optical absorption spectra. We find that including nuclear quantum effects in the sampling of chromophore-solvent configurations using ab initio path integral molecular dynamics simulations leads to improved spectral shapes through three mechanisms. The three mechanisms that lead to line shape broadening and a better description of the high-energy tail are softening of heavy atom bonds in the chromophore that couple to the optically bright state, widening the distribution of vertical excitation energies from more diverse solvation environments, and redistributing spectral weight from the 0-0 vibronic transition to higher energy vibronic transitions when computing the Franck-Condon spectrum in a frozen solvent pocket. The absorption spectra computed using the combined ensemble plus zero-temperature Franck-Condon approach yield significant improvements in spectral shape and width compared to the spectra computed with the ensemble approach. Using the combined approach with configurations sampled from path integral molecular dynamics trajectories presents a significant step forward in accurately modeling the absorption spectra of aqueously solvated chromophores. [ABSTRACT FROM AUTHOR]

The article presents a study which demonstrated the need for conformational ensembles to understand function, using the enzyme ketosteroid isomerase (KSI) as an example. Topics discussed include KSI structure formation and the need for ensemble function, KSI expression and purification, and protein crystallization and x-ray data collection.

Keywords: light-regulation; non-canonical amino acids; optogenetics; photopharmacology; photoxenoprotein engineering; proteins EN light-regulation non-canonical amino acids optogenetics photopharmacology photoxenoprotein engineering proteins 573 613 41 04/12/22 20220401 NES 220401 Introduction Light plays a decisive role for most forms of life as it stimulates various essential processes in organisms. In many cases, the photoswitchable ligand is bioconjugated to a self-labeling protein tag such as the SNAP tag, which is genetically fused with either the target protein or an auxiliary protein interacting with the target protein ([174]; [83]). (B) The four main strategies of hybrid protein optogenetics including transduction of conformational changes, steric decaging of proteins or peptides, homotypic association/dissociation and heterotypic association/dissociation of heterocomplexes or split proteins. A generally applicable protocol has hitherto not been established due to the fact that each protein is individual; a strategy, which facilitates a high LRF for one protein, might not be able to attain photocontrol of another protein at all. The target proteins must further be specific for the cells that are the cause of the disease, e.g., overproduced proteins in tumor cells or proteins that solely exist in bacteria. [Extracted from the article]

The article presents a study that explores tracking the Adenosine triphosphate (ATP)-binding response in adenylate kinase in real time. It mentions about time-resolved x-ray solution scattering (TR-XSS) to visualize structural changes in the Escherichia coli adenylate kinase (AdK) enzyme upon laser-induced activation of a protected ATP substrate.

A detailed theoretical analysis, based on extensive ab initio second-order approximate coupled cluster calculations, has been performed for the S1 potential energy surface (PES) of four photoactive yellow protein (PYP) chromophore derivatives that are hydrogen bonded with two water molecules and differ merely in the carbonyl substituent. The main focus is put on contrasting the isomerization properties of these four species in the S1 excited state, related to torsion around the chromophore's single and double carbon-carbon bonds. The analysis provides evidence of the different isomerization behavior of these four chromophore complexes, which relates to the difference in their carbonyl substituents. While a stable double-bond torsion pathway exists on the S1 PES of the chromophores bearing the -O-CH3 and -NH2 substituents, this is not the case for the -S-CH3 and -CH3 substituted species. The presence of the -S-CH3 group leads to a strong instability of the chromophore with respect to the single-bond twist, whereas in the case of the -CH3 substituent a crossing of the S1 and S2 PESs occurs, which perturbs the pathway. Based on this analysis, the key factors that support the double-bond torsion have been identified. These are (i) the hydrogen bonds at the phenolic oxygen of the chromophore, (ii) the weak electron-acceptor character of the carbonyl group, and (iii) the ethylene-like pattern of the torsion in the beginning of the process. Our results suggest that the interplay between these factors determines the chromophore's isomerization in the solvent environment and in the native PYP environment. [ABSTRACT FROM AUTHOR]

A combined quantum mechanical/molecular mechanical/continuum (QM/MM/C) style second order Møller-Plesset perturbation theory (MP2) method that incorporates induced dipole polarizable force field and induced surface charge continuum solvation model is established. The Z-vector method is modified to include induced dipoles and induced surface charges to determine the MP2 response density matrix, which can be used to evaluate MP2 properties. In particular, analytic nuclear gradient is derived and implemented for this method. Using the Assisted Model Building with Energy Refinement induced dipole polarizable protein force field, the QM/MM/C style MP2 method is used to study the hydrogen bonding distances and strengths of the photoactive yellow protein chromopore in the wild type and the Glu46Gln mutant. [ABSTRACT FROM AUTHOR]

The article presents a study comparing the crystallographic diffraction data collected by serial femtosecond and serial synchrotron crystallography for the proteins myoglobin and fluoroacetate dehalogenase. It discusses the two protein systems exploited to evaluate the differences in quality, radiation damage as a limiting factor in achieving high-resolution structures at cryogenic temperatures, and protein expression and crystallization.

The structure of experimentally designed solar cells was optimized in terms of the photoactive layer thickness for both organic bulk heterojunction and hybrid perovskite solar cells. The photoactive layer thickness had a totally different behavior on the performance of the organic and hybrid solar cells. Analysis of the optical parameters using transfer matrix modeling within the Maxwell–Garnett effective refractive index model shows that light absorbance and exciton generation rate in the photoactive layer can be used to optimize the thickness range of the photoactive layer. Complete agreement between experimental and simulated data for solar cells with photoactive materials that have very different natures proves the validity of the proposed modeling method. The proposed simple method which is not time-consuming to implement permits to obtain a preliminary assessment of the reasonable range of layer thickness that will be needed for designing experimental samples. [ABSTRACT FROM AUTHOR]

This perspective article highlights the challenges in the theoretical description of photoreceptor proteins using multiscale modeling, as discussed at the CECAM workshop in Tel Aviv, Israel. The participants have identified grand challenges and discussed the development of new tools to address them. Recent progress in understanding representative proteins such as green fluorescent protein, photoactive yellow protein, phytochrome, and rhodopsin is presented, along with methodological developments. [ABSTRACT FROM AUTHOR]

A combined quantum mechanical/molecular mechanical/continuum (QM/MMpol/C) style method is developed for time-dependent density functional theory (TDDFT, including long-range corrected TDDFT) method, induced dipole polarizable force field, and induced surface charge continuum model. Induced dipoles and induced charges are included in the TDDFT equations to solve for the transition energies, relaxed density, and transition density. Analytic gradient is derived and implemented for geometry optimization and molecular dynamics simulation. QM/MMpol/C style DFT and TDDFT methods are used to study the hydrogen bonding of the photoactive yellow protein chromopore in ground state and excited state. [ABSTRACT FROM AUTHOR]

Although widely used in the detection and characterization of protein–protein interactions, Y2H screening has been under-used for the engineering of new optogenetic tools or the improvement of existing tools. Here we explore the feasibility of using Y2H selection and screening to evaluate libraries of photoswitchable protein–protein interactions. We targeted the interaction between circularly permuted photoactive yellow protein (cPYP) and its binding partner binder of PYP dark-state (BoPD) by mutating a set of four surface residues of cPYP that contribute to the binding interface. A library of ~ 10,000 variants was expressed in yeast together with BoPD in a Y2H format. An initial selection for the cPYP/BoPD interaction was performed using a range of concentrations of the cPYP chromophore. As expected, the majority (> 90% of cPYP variants) no longer bound to BoPD. Replica plating was then used to evaluate the photoswitchability of the surviving clones. Photoswitchable cPYP variants with BoPD affinities equal to, or higher than, native cPYP were recovered in addition to variants with altered photocycles and binders that interacted with BoPD as apo-proteins. Y2H results reflected protein–protein interaction affinity, expression, photoswitchability, and chromophore uptake, and correlated well with results obtained both in vitro and in mammalian cells. Thus, by systematic variation of selection parameters, Y2H screens can be effectively used to generate new optogenetic tools for controlling protein–protein interactions for use in diverse settings. Unlabelled Image • A library of ~ 10,000 variants of photoactive yellow protein (PYP) was evaluated for binding to a partner protein Binder of PYP-Dark (BoPD) using a yeast two-hybrid approach. • By varying parameters of the two-hybrid selection, distinct photoswitchable behaviors were recovered including PYP variants with altered affinity, kinetics, and apo-state binding. • Phenotypes observed in yeast two-hybrid assays correlate well with results obtained in vitro and in mammalian cells. [ABSTRACT FROM AUTHOR]

PYP-phytochrome related (Ppr) protein contains the two light sensor domains, photoactive yellow protein (PYP) and bacteriophytochrome (Bph), which mainly absorb blue and red light by the chromophores of pcoumaric acid (pCA) and biliverdin (BV), respectively. As a result, Ppr has the ability to photoactivate both domains together or separately. We investigated the photoreaction of each photosensor domain under different light irradiation conditions and clarified the inter-dependency between these domains. Within the first 10 s of blue light illumination, Ppr (Holo-Holo-Ppr) accompanied by both pCA and BV demonstrated spectrum changes reflecting PYPL accumulation, which can also be observed in Ppr containing only pCA (Holo-Apo-Ppr), and a fragment of Ppr lacking the C-terminal Bph domain. Although Holo-Apo-Ppr showed PYPL as a major photoproduct under blue light, as seen in the Bph-truncated Ppr, the equilibrium in the Holo-Holo-Ppr was shifted from PYPL to PYPM as the reaction progresses under blue light. Concomitantly, the spectrum of Bph exhibited subtle but distinguishable alteration. Together with the fact, it can be proposed that Bph with BV influences the photoreaction of PYP in Ppr, and vice versa. SAXS measurements revealed substantial tertiary structure changes in Holo-Holo-Ppr under continuous blue light irradiation within the first 5 min time domain. Interestingly, the changes in tertiary structure were partially suppressed by photoactivation of the Bph domain. These observations indicate that the photoreactions of the PYP and Bph domains are coupled with each other, and that the interplay realizes the structural switch, which might be involved in downstream signal transduction. [ABSTRACT FROM AUTHOR]

Labelling technologies developed over the past few years have changed the way of looking at biomolecules and have made a considerable contribution to our understanding of the functions and regulation of dynamic biological processes. One of the robust technologies employed to image proteins in a cellular environment is based on the use of chemical tags and their fluorescent probes, which provides flexibility in developing probes with a wide range of synthetic fluorophores. A variety of chemical tags, ranging from short amino acid sequences to small proteins, have been employed to generate protein-labelling systems. One such chemical tag is the photoactive yellow protein (PYP)-tag, which is a small bacterial protein, developed for the selective labelling and imaging of proteins. Herein, we briefly discuss the protein-labelling system developed based on PYP-tag technology, with a focus on the design strategy for PYP-tag labelling probes and their applications in protein imaging. [ABSTRACT FROM AUTHOR]

In this paper, the photocycle of the dried photoactive yellow protein film has been investigated in different humidity environments, in order to characterize its nonlinear optical properties for possible integrated optical applications. The light-induced spectral changes of the protein films were monitored by an optical multichannel analyser set-up, while the accompanying refractive index changes were measured with the optical waveguide lightmode spectroscopy method. To determine the number and kinetics of spectral intermediates in the photocycle, the absorption kinetic data were analysed by singular value decomposition and multiexponential fitting methods, whose results were used in a subsequent step of fitting a photocycle model to the data. The absorption signals of the films were found to be in strong correlation with the measured light-induced refractive index changes, whose size and kinetics imply that photoactive yellow protein may be a good alternative for utilization as an active nonlinear optical material in future integrated optical applications. [ABSTRACT FROM AUTHOR]

Based on a bacterial blue light photoreceptor, photoactive yellow protein (PYP), we have developed a photoresponsive protein module, Hpg5‐PYP, bearing five alkynyl groups as reactive sites. We constructed a yellow‐colored photoresponsive protein–polymer conjugate by using covalently bonded Hpg5‐PYP with poly(N,N‐dimethylacrylamide‐co‐2‐hydroxy‐3‐azidopropyl methacrylate) (DMA‐HAZPMA copolymer). Hpg5‐PYP retained photoreactivity towards blue‐light irradiation in the conjugate, and the conjugate showed a photoresponsive viscosity change upon irradiation with blue light at 25 °C. In contrast, a mixture of Hpg5‐PYP and DMA‐HAZPMA copolymer prepared for reference did not show any dependency of the viscosity on light irradiation, indicating the importance of crosslinking. [ABSTRACT FROM AUTHOR]

The article reports that fifth-order time-domain Raman spectroscopy of photoactive yellow protein (PYP), with the aim to visualize vibrational coupling in its excited state. The unveiled coupling suggests the importance of the low-frequency vibrational motion in the primary photoreaction of PYP highlighting the unique capability of this spectroscopic approach for studying ultrafast reaction dynamics.

Photoactive proteins that efficiently and selectively transfer light energy into a physical response are ubiquitous in nature. The small molecule chromophores that lie at the heart of these processes often exist as closed-shell anions following deprotonation in proton-transfer reactions. This review highlights the important role that anion photoelectron spectroscopy, combined with computational chemistry calculations, is playing in improving our understanding of the electronic structure and relaxation dynamics of these protein chromophores. We discuss key aspects of anion photoelectron spectroscopy. We then review recent anion photoelectron spectroscopy studies of the deprotonated chromophore anions found in green fluorescent protein (GFP), photoactive yellow protein (PYP) and the deprotonated luciferin anion found in the luciferase enzyme. [ABSTRACT FROM AUTHOR]

Normal mode analysis provides a powerful tool in biophysical computations. Particularly, we shed light on its application to protein properties because they directly lead to biological functions. As a result of normal mode analysis, the protein motion is represented as a linear combination of mutually independent normal mode vectors. It has been widely accepted that the large amplitude motions throughout the entire protein molecule can be well described with a few low-frequency normal modes. Furthermore, it is possible to represent the effect of external perturbations, e.g., ligand binding, hydrostatic pressure, as the shifts of normal mode variables. Making use of this advantage, we are able to explore mechanical properties of proteins such as Young’s modulus and compressibility. Within thermally fluctuating protein molecules under physiological conditions, tightly packed amino acid residues interact with each other through heat and energy exchanges. Since the structure and dynamics of protein molecules are highly anisotropic, the flow of energy and heat should also be anisotropic. Based on the harmonic approximation of the heat current operator, it is possible to analyze the communication map of a protein molecule. By using this method, the energy transfer pathways of photoactive yellow protein were calculated. It turned out that these pathways are similar to those obtained via the Green-Kubo formalism with equilibrium molecular dynamics simulations, indicating that normal mode analysis captures the intrinsic nature of the transport properties of proteins. [ABSTRACT FROM AUTHOR]

Molecular and supramolecular‐based systems and materials that can perform predetermined functions in response to light stimulation have been extensively studied in the past three decades. Their investigation continues to be a highly stimulating topic of chemical research, not only because of the inherent scientific value related to a bottom‐up approach to functional nanostructures, but also for the prospective applications in diverse fields of technology and medicine. Light is an important tool in this context, as it can be conveniently used both for supplying energy to the system and for probing its states and transformations. In this microreview we recall some basic aspects of light‐induced processes in (supra)molecular assemblies, and discuss their exploitation to implement novel functionalities with nanostructured devices, machines and materials. To this aim we illustrate a few examples from our own recent work, which are meant to illustrate the trends of current research in the field. A bright future: the combination of the concepts and tools of photophysics and photochemistry with appropriately designed molecular and supramolecular systems can lead to the realization of nanoscale devices, machines and materials that can execute sophisticated functions in response to light stimulation. [ABSTRACT FROM AUTHOR]

Abstract The direct conversion of sunlight into chemical energy via photosynthesis is a unique capability of plants and some bacterial species. Aimed at mimicking this energy conversion process, the combination of inorganic substrates and organic photoactive proteins into an artificial biohybrid system is of a great interest for artificial bio-photovoltaic applications. It also allows to better understand charge transfer processes involved in the photosynthetic chain. In this work, single layer graphene (SLG) and multilayer graphene (MLG) electrodes are used as a platform for the immobilization of reaction centers (RCs) from purple bacteria Rhodobacter sphaeroides , a protein complex responsible for the generation of photo-excited charges. Electrochemical experiments with graphene electrodes and redox molecules reveal fundamental differences in the charge transfer processes for SLG and MLG films. We demonstrate that both graphene-based materials enable the immobilization of RCs without loss of functionality, attested by a photocurrent generation under illumination with IR-light at a wavelength of 870 nm. Furthermore, we report on the dependence of the generated photocurrent on the applied bias voltage, as well as on the presence of charge mediators in the surrounding electrolyte. This work demonstrates that SLG and MLG are a suitable platform for RC immobilization and subsequent photocurrent generation, suggesting a promising potential for graphene-based materials in bio-photovoltaics. Graphical abstract Unlabelled Image Highlights • Charge transfer processes of single and multilayer graphene electrodes • Immobilization of bacterial reaction centers on graphene electrodes • Comparison of photovoltaic performance of reaction centers on single and multilayer graphene electrodes [ABSTRACT FROM AUTHOR]

Rational design of photoreagents with systematic modifications of their structures can provide valuable information for a better understanding of the protein photocleavage mechanism by these reagents. Variation of the length of the linker connecting the photoactive moiety with the protein anchoring-group allowed us to investigate the control of the protein photocleavage site. A series of new photochemical reagents (PMA-1A, PMA-2A and PMA-3A) with increasing chain lengths is examined in the current study. Using avidin as a model system, we examined the interaction of these probes by UV–Vis, fluorescence spectroscopic methods, photocleavage and computational docking studies. Hypochromism of the absorption spectrum was observed for the binding of these new photochemical reagents with estimated binding constants (K b ) of 6.2 × 10 5 , 6.7 × 10 5 and 4.6 × 10 5  M −1 , respectively. No significant changes of Stern-Volmer quenching constant (K sv ) with Co(NH 3 ) 6 Cl 3 has been noted and the data indicated that the probes bind near the surface of the protein with sufficient exposure to the solvent. Photoexcitation of the probe-avidin complex, in the presence of Co(NH 3 ) 6 Cl 3 , resulted in protein fragmentation, and the cleavage yield decreased with the increase in the linker length, and paralleled with the observed K sv values. Amino acid sequencing of the photofragments indicated that avidin is cleaved between Thr77 and Val78, as a major cleavage site for all the three photoreagents. This site is proximate to the biotin binding site on avidin, and molecular docking studies indicated that the H-bonding interactions between the polar end-group of the photoreagents and hydrophilic amino acids of avidin were important in positioning the reagent on the protein. The major cleavage site, at residues 77–78, was within 5 Å of the pyrenyl moiety of the probe, and hence, molecular tuning of the linker provided a simple approach to position the photoreagent along the potential photocleavage site. [ABSTRACT FROM AUTHOR]

Photoactive yellow protein (PYP), from the phototrophic bacterium Halorhodospira halophila, is a small water-soluble photoreceptor protein and contains p-coumaric acid (pCA) as a chromophore. PYP has been an attractive model for studying the physical chemistry of protein active sites. Here, we explore how Raman optical activity (ROA) can be used to extract quantitative information on distortions of the pCA chromophore at the active site in PYP. We use 13C8-pCA to assign an intense signal at 826 cm-1 in the ROA spectrum of PYP to a hydrogen out-of-plane vibration of the ethylenic moiety of the chromophore. Quantum-chemical calculations based on density functional theory demonstrate that the sign of this ROA band reports the direction of the distortion in the dihedral angle about the ethylenic C=C bond, while its amplitude is proportional to the dihedral angle. These results document the ability of ROA to quantify structural deformations of a cofactor molecule embedded in a protein moiety. [ABSTRACT FROM AUTHOR]

Semiconductor photocatalysis using single or modified semiconductors and heterojunction-type composites is limited due to lower visible light response, rapid recombination of photogenerated charge carriers and low redox potentials of photoactive materials. Recently the coordinated applications of two or more semiconductors are becoming popular, an idea borrowed from the thermodynamically uphill driven tandem Z-scheme concept used for photoelectrochemical splitting of water involving two different semiconductors with improved overall visible light response. Consequently, in thermodynamically downhill photocatalytic reactions for pollutant degradation involving dual semiconductors as well the word “Z-scheme” is being used to explain the observed efficient electron-hole separation mechanism. Whereas the Z-scheme classically represents only thermodynamically uphill photosynthetic reactions (ΔG > 0), the photocatalytic degradation of pollutants, on the other hand, are thermodynamically downhill reactions (ΔG < 0), and this usage is creating a misconception and ambiguity in the field of photocatalysis. Therefore, herein we discuss the principles of the often called ‘Z-scheme photosystems’ involving (i) shuttle redox-mediators, (ii) dual semiconductors photocatalysts with solid state electron mediators and (iii) redox-mediator-free direct systems. The review relevantly summarizes and discusses various representative dual semiconductor photocatalytic systems with solid state mediators as well as non-mediated direct dual semiconductor photocatalysts studied for the degradation of pollutants under visible light irradiation. The future requirements for the development of an efficient visible light driven redox-mediator-free and durable direct dual semiconductor photocatalytic system for the degradation of pollutants is also presented. [ABSTRACT FROM AUTHOR]

Photoactive yellow protein (PYP) induces negative phototaxis in Halorhodospira halophila via photoactivation triggered by light-mediated chromophore isomerization. Chromophore isomerization proceeds via a volume-conserving isomerization mechanism due to the hydrogen-bond network and steric constraints inside the protein, and causes significant conformational changes accompanied by N-terminal protrusion. However, it is unclear how the structural change of the chromophore affects the remote N-terminal domain. To understand photocycle-related structural changes, we investigated the structural aspect of chromophore removal in PYP because it possesses a disrupted hydrogen-bond network similar to that in photocycle intermediates. A comparison of the structural aspects with those observed in the photocycle would give a clue related to the structural change mechanism in the photocycle. Chromophore removal effects were assessed via UV-vis spectroscopy, circular dichroism, and X-ray solution scattering. Molecular shape reconstruction and an experiment-restrained rigid-body molecular dynamics simulation based on the scattering data were performed to determine protein shape, size, and conformational changes upon PYP bleaching. Data show that chromophore removal disrupted the holo-PYP structure, resulting in a small N-terminal protrusion, but the extent of conformational changes was markedly less than those in the photocycle. This indicates that disruption of the hydrogen-bond network alone in bleached PYP does not induce the large conformational change observed in the photocycle, which thus must result from the organized structural transition around the chromophore triggered by chromophore photoisomerization along with disruption of the hydrogen-bond network between the chromophore and the PYP core. [ABSTRACT FROM AUTHOR]

Organic polymer solar cells (PSCs) have attracted increasing attention due to light weight, low cost, flexibility and roll-to-roll manufacturing. However, the limited light harvest range of the photoactive layer greatly restrains the power conversion efficiency (PCE) enhancement. In order to expand the light absorption range and further enhance the PCE of the PSCs, tandem structures have been designed and demonstrated. In tandem solar cell, the intermediate layer (IML) plays a critical role in physically and electrically connection of the two subcells. Herein, we apply titanium (diisopropoxide) bis(2,4-pentanedionate) (TIPD) as both electrode modification layer and intermediate layer to investigate the feasibility in inverted tandem polymer solar cells. The same photoactive layers of PTB7-Th:PC71BM are adopted in both front and rear subcells to simplify the evaluation of effectiveness of TIPD layer in tandem structures. By modulating the treatment condition of IML and the thickness of photoactive layer, efficient inverted tandem PSCs have been achieved with minimized voltage loss and excellent charge transportation, giving a best Voc of 1.54 V, which is almost two times that of the single bulk heterojunction (BHJ)-PSC (0.78 V) and an enhanced PCE up to 8.11%. [ABSTRACT FROM AUTHOR]

Interfacial materials are essential to the performance and stability of polymer solar cells (PSCs). Herein, solution-processed zirconium(IV) isopropoxide (Zr[OCH(CH3)2]2, ZrIPO) has been employed as an efficient cathode buffer layer between the Al cathode and photoactive layer. The ZrIPO buffer layer is prepared simply via spin-coating its isopropanol solution on the photoactive layer at room temperature without any post-treatment. When using ZrIPO/Al instead of the traditionally used Ca/Al cathode in PSCs, the short-circuit current density (Jsc) is significantly improved and the series resistance of the device is decreased. The power conversion efficiency (PCE) of the P3HT:PCBM-based device with ZrIPO buffer layer reaches 4.47% under the illumination of AM1.5G, 100 mW/cm2. A better performance with PCE of 8.07% is achieved when a low bandgap polymer PBDTBDD is selected as donor material. The results indicate that ZrIPO is a promising electron collection material as a substitute of the traditional low-work-function cathode for high performance PSCs. [ABSTRACT FROM AUTHOR]

We have implemented a linear-scaling divide-and-conquer (DC)-based higher-order coupled-cluster (CC) and Møller-Plesset perturbation theories (MPPT) as well as their combinations automatically by means of the tensor contraction engine, which is a computerized symbolic algebra system. The DC-based energy expressions of the standard CC and MPPT methods and the CC methods augmented with a perturbation correction were proposed for up to high excitation orders [e.g., CCSDTQ, MP4, and CCSD(2)TQ]. The numerical assessment for hydrogen halide chains, polyene chains, and first coordination sphere (C1) model of photoactive yellow protein has revealed that the DC-based correlation methods provide reliable correlation energies with significantly less computational cost than that of the conventional implementations. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

To elucidate the role of internal proton transfer in the photodynamics of photoactive yellow protein (PYP), the photocycle of the Glu46Gln mutant of PYP (E46Q-PYP) is investigated by transient grating (TG) spectroscopy. Compared with wild-type PYP (wt-PYP), the first spectrally blue-shifted intermediate of E46Q-PYP is formed more slowly, which is consistent with the absence of direct protonation from Glu46 residue, if the parallel kinetic model for wt-PYP is invoked. The smaller conformational change in E46Q-PYP, as manifested by the smaller change in the diffusion coefficient, mainly arises from the relatively larger volume of the ground state E46Q-PYP than wt-PYP rather than from the smaller volume of the pB state. [ABSTRACT FROM AUTHOR]

Contents937I.937II.938III.939IV.943V.947VI.948948References949 Summary: The orange carotenoid protein (OCP) is a water‐soluble, photoactive protein involved in thermal dissipation of excess energy absorbed by the light‐harvesting phycobilisomes (PBS) in cyanobacteria. The OCP is structurally and functionally modular, consisting of a sensor domain, an effector domain and a keto‐carotenoid. On photoactivation, the OCP converts from a stable orange form, OCPO, to a red form, OCPR. Activation is accompanied by a translocation of the carotenoid deeper into the effector domain. The increasing availability of cyanobacterial genomes has enabled the identification of new OCP families (OCP1, OCP2, OCPX). The fluorescence recovery protein (FRP) detaches OCP1 from the PBS core, accelerating its back‐conversion to OCPO; by contrast, other OCP families are not regulated by FRP. N‐terminal domain homologs, the helical carotenoid proteins (HCPs), have been found among diverse cyanobacteria, occurring as multiple paralogous groups, with two representatives exhibiting strong singlet oxygen (1O2) quenching (HCP2, HCP3) and another capable of dissipating PBS excitation (HCP4). Crystal structures are presently available for OCP1 and HCP1, and models of other HCP subtypes can be readily produced as a result of strong sequence conservation, providing new insights into the determinants of carotenoid binding and 1O2 quenching. [ABSTRACT FROM AUTHOR]

Photo absorption properties of p-coumaric acid, the chromophore of photoactive yellow protein, in aqueous solution were investigated by means of reference interaction site model self-consistent field with spatial electron density distribution (RISM-SCF-SEDD) method. RISM-SCF-SEDD is a combination methodology of electronic structure theory and statistical mechanics for molecular liquids. Here, time-dependent density functional theory was coupled with RISM equation to study the electronic structure of p-coumaric acid in aqueous system. Excitation energies of the chromophore in its neutral, two monoanionic and dianionic forms were computed to elucidate the effect of the deprotonation and solvation on the spectroscopic properties. We found that solvation strongly affects the excitation character of the chromophore, especially for phenolate anion and dianion. The free energy difference among the four protonation states is also discussed. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Using various spectroscopic techniques such as UV-visible spectroscopy, circular dichroism spectroscopy, NMR spectroscopy, small-angle X-ray scattering, transient grating, and transient absorption techniques, we investigated how cell-mimetic environments made by crowding influence the photocycle of photoactive yellow protein (PYP) in terms of the molecular volume change and kinetics. Upon addition of molecular crowding agents, the ratio of the diffusion coefficient of the blue-shifted intermediate (pB) to that of the ground species (pG) significantly changes from 0.92 and approaches 1.0. This result indicates that the molecular volume change accompanied by the photocycle of PYP in molecularly crowded environments is much smaller than that which occurs in vitro and that the pB intermediate under crowded environments favors a compact conformation due to the excluded volume effect. The kinetics of the photocycle of PYP in cell-mimetic environments is greatly decelerated by the dehydration, owing to the interaction between the protein and small crowding agents, but is barely affected by the excluded volume effect. The results lead to the inference that the signaling transducer of PYP may not necessarily utilize the conformational change of PYP to sense the signaling state. [ABSTRACT FROM AUTHOR]

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems. [ABSTRACT FROM AUTHOR]

Ultrafast X-ray crystallography of the photoactive yellow protein with femtosecond delays using an X-ray free electron laser has successfully probed the dynamics of an early Franck-Condon species. The femtosecond pump-probe application of protein crystallography represents a new experimental regime that provides an X-ray structural probe for coherent processes that were previously accessible primarily using ultrafast spectroscopy. We address how the optical regime of the visible pump, that is necessary to successfully resolve ultrafast structural differences, affects the motions that are measured using the technique. The sub-picosecond photochemical dynamics in PYP involves evolution of a mixture of electronic ground and excited state populations. Additional to photoisomerisation that is considered to proceed through activated barrier crossing, within the dephasing time structural motion include vibrational coherence arising from excited states, the ground state and a ground state intermediate under experimental conditions used for ultrafast crystallography. Intense optical pulses are required to convert population levels in PYP crystals that allow detection by X-ray crystallography, but the compromise currently needed for the optical bandwidth and power has consequences with regard to the contributions to the motions that are experimentally measured with femtosecond delays. We briefly review the ultrafast spectroscopy literature of the primary photoreactions of PYP and discuss relevant physical models taken from coherent control and femtosecond coherence spectroscopy literature that address both the population transfer as well as the vibrational coherences. We apply linear response theory, with the additional use of a high power approximation, of on-resonance impulsive vibrational coherence in the ground state and the non-impulsive coherence in the excited state and discuss experimental approaches to manipulate the coherence contributions. The results are generalised and extended to discuss the future capabilities of high repetition rate X-ray free electron laser instruments providing enhanced sensitivity to perform the crystallographic equivalent of an impulsive Raman measurement of vibrational coherence. [ABSTRACT FROM PUBLISHER]

Because arginine residues in proteins are expected to be in their protonated form almost without exception, reports demonstrating that a protein arginine residue is charge-neutral are rare and potentially controversial. Herein, we present a 13C-detected NMR experiment for probing individual arginine residues in proteins notwithstanding the presence of chemical and conformational exchange effects. In the experiment, the 15Nη and 15Nϵ chemical shifts of an arginine head group are correlated with that of the directly attached 13Cζ. In the resulting spectrum, the number of protons in the arginine head group can be obtained directly from the 15N-1H scalar coupling splitting pattern. We applied this method to unambiguously determine the ionization state of the R52 side chain in the photoactive yellow protein from Halorhodospira halophila. Although only three Hη atoms were previously identified by neutron crystallography, we show that R52 is predominantly protonated in solution. [ABSTRACT FROM AUTHOR]

There has been considerable debate on the existence of a low-barrier hydrogen bond (LBHB) in the photoactive yellow protein (PYP). The debate was initially triggered by the neutron diffraction study of Yamaguchi et al. ( Proc. Natl. Acad. Sci., U. S. A., 2009, 106, 440−444 ) who suggested a model in which a neutral Arg52 residue triggers the formation of the LBHB in PYP. Here, we present an alternative model that is consistent within the error margins of the Yamaguchi structure factors. The model explains an increased hydrogen bond length without nuclear quantum effects and for a protonated Arg52. We tested both models by calculations under crystal, solution, and vacuum conditions. Contrary to the common assumption in the field, we found that a single PYP in vacuum does not provide an accurate description of the crystal conditions but instead introduces strong artifacts, which favor a LBHB and a large1H NMR chemical shift. Our model of the crystal environment was found to stabilize the two Arg52 hydrogen bonds and crystal water positions for the protonated Arg52 residue in free MD simulations and predicted an Arg52 pK aupshift with respect to PYP in solution. The crystal and solution environments resulted in almost identical1H chemical shifts that agree with NMR solution data. We also calculated the effect of the Arg52 protonation state on the LBHB in 3D nuclear equilibrium density calculations. Only the charged crystal structure in vacuum supports a LBHB if Arg52 is neutral in PYP at the previously reported level of theory ( J. Am. Chem. Soc., 2014, 136, 3542−3552 ). We attribute the anomalies in the interpretation of the neutron data to a shift of the potential minimum, which does not involve nuclear quantum effects and is transferable beyond the Yamaguchi structure. [ABSTRACT FROM AUTHOR]

A modulable and simple approach towards heteroleptic ruthenium(ii) complexes of amidine-based polypyridine ligands is presented. New complexes 1 and 2 ([(terpyridine)Ru(terpyridine-C6H4-C(NR)(NHR))]2+ with R = propyl and R = phenyl derivatives, respectively) were characterized by NMR spectroscopy in solution and by X-ray diffraction, which confirmed the obtention of the (E) stereoisomer alone. Depending on the bulkiness of the R-substituents introduced on the amidine moiety, rotational isomerism around the C–N bond could be observed at r.t. Spectroscopic and electrochemical studies showed that the nature of the R-substituents introduced on the amidine moiety can significantly influence the redox and ground-state acido-basic properties of the complexes, while maintaining their electronic features. This particular tunability of polytopic 4′-(amidinato)-terpyridines offers an interesting perspective for photoactive units in larger multi-functional arrays. [ABSTRACT FROM AUTHOR]

Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag), by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure. [ABSTRACT FROM AUTHOR]

A novel metallocyclodextrins polymer was synthesized as artificial polymeric receptor. The polymer showed excellent adsorption stability on the surface of glassy carbon electrode. More interestingly, the receptor could be prepared by using molecular imprinting technique conveniently and the resulting polymer template exhibited excellent selectivity. These attractive characteristics will be significant for constructing ultrasensitive ECL sensors. [ABSTRACT FROM AUTHOR]