Unraveling the Intrinsic Color of Chlorophyll

The exact color of light absorbed by chlorophyll (Chl) pigments, the light‐harvesters in photosynthesis, is tuned by the protein microenvironment, but without knowledge of the intrinsic color of Chl it remains unclear how large this effect is. Experimental first absorption energies of Chl a and b isolated in vacuo and tagged with quaternary ammonium cations are reported. The energies are largely insensitive to details of the tag structure, a finding supported by first‐principles calculations using time‐dependent density functional theory. Absorption is significantly blue‐shifted compared to that of Chl‐containing proteins (by 30–70 nm). A single red‐shifting perturbation, such as axial ligation or the protein medium, is insufficient to account even for the smallest shift; the largest requires pigment–pigment interactions.     

On the Exciton Coupling between Two Chlorophyll Pigments in the Absence of a Protein Environment: Intrinsic Effects Revealed by Theory and Experiment

Exciton coupling between two or more chlorophyll (Chl) pigments is a key mechanism associated with the color tuning of photosynthetic proteins but it is difficult to disentangle this effect from shifts that are due to the protein microenvironment. Herein, we report the formation of the simplest coupled system, the Chl a dimer, tagged with a quaternary ammonium ion by electrospray ionization. Based on action spectroscopic studies in vacuo, the dimer complexes were found to absorb 50–70 meV to the red of the monomers under the same conditions. First‐principles calculations predict shifts that somewhat depend on the relative orientation of the two Chl units, namely 50 and 30 meV for structures where the Chl rings are stacked and unstacked, respectively. Our work demonstrates that Chl association alone can produce a large portion of the color shift observed in photosynthetic macromolecular assemblies.     

Color Tuning in rhodopsins: the origin of the spectral shift between the chloride-bound and anion-free forms of halorhodopsin

Detailed knowledge of the molecular mechanisms that control the spectral properties in the rhodopsin protein family is important for understanding the functions of these photoreceptors and for the rational design of artificial photosensitive proteins. Here we used a high-level ab initio QM/MM method to investigate the mechanism of spectral tuning in the chloride-bound and anion-free forms of halorhodopsin from Natronobacterium pharaonis (phR) and the interprotein spectral shift between them. We demonstrate that the chloride ion tunes the spectral properties of phR via two distinct mechanisms: (i) electrostatic interaction with the chromophore, which results in a 95 nm difference between the absorption maxima of the two forms, and (ii) induction of a structural reorganization in the protein, which changes the positions of charged and polar residues and reduces this difference to 29 nm. The present study expands our knowledge concerning the role of the reorganization of the internal H-bond network for color tuning in general and provides a detailed investigation of the tuning mechanism in phR in particular.     

Color tuning in rhodopsins: the mechanism for the spectral shift between bacteriorhodopsin and sensory rhodopsin II

The mechanism of color tuning in the rhodopsin family of proteins has been studied by comparing the optical properties of the light-driven proton pump bacteriorhodopsin (bR) and the light detector sensory rhodopsin II (sRII). Despite a high structural similarity, the maximal absorption is blue-shifted from 568 nm in bR to 497 nm in sRII. The molecular mechanism of this shift is still a matter of debate, and its clarification sheds light onto the general mechanisms of color tuning in retinal proteins. The calculations employ a combined quantum mechanical/molecular mechanical (QM/MM) technique, using a DFT-based method for ground state properties and the semiempirical OM2/MRCI method and ab initio SORCI method for excited state calculations. The high efficiency of the methodology has allowed us to study a wide variety of aspects including dynamical effects. The absorption shift as well as various mutation experiments and vibrational properties have been successfully reproduced. Our results indicate that several sources contribute to the spectral shift between bR and sRII. The main factors are the counterion region at the extracellular side of retinal and the amino acid composition of the binding pocket. Our analysis allows a distinction and identification of the different effects in detail and leads to a clear picture of the mechanism of color tuning, which is in good agreement with available experimental data.     

The Lack of Lutein Accelerates the Extent of Light‐induced Bleaching of Photosynthetic Pigments in Thylakoid Membranes of Arabidopsis thaliana

The high light‐induced bleaching of photosynthetic pigments and the degradation of proteins of light‐harvesting complexes of PSI and PSII were investigated in isolated thylakoid membranes of Arabidopsis thaliana, wt and lutein‐deficient mutant lut2, with the aim of unraveling the role of lutein for the degree of bleaching and degradation. By the means of absorption spectroscopy and western blot analysis, we show that the lack of lutein leads to a higher extent of pigment photobleaching and protein degradation in mutant thylakoid membranes in comparison with wt. The highest extent of bleaching is suffered by chlorophyll a and carotenoids, while chlorophyll b is bleached in lut2 thylakoids during long periods at high illumination. The high light‐induced degradation of Lhca1, Lhcb2 proteins and PsbS was followed and it is shown that Lhca1 is more damaged than Lhcb2. The degradation of analyzed proteins is more pronounced in lut2 mutant thylakoid membranes. The lack of lutein influences the high light‐induced alterations in organization of pigment–protein complexes as revealed by 77 K fluorescence.     

Tuning the Protein‐Induced Absorption Shifts of Retinal in Engineered Rhodopsin Mimics

Rational design of light‐capturing properties requires understanding the molecular and electronic structure of chromophores in their native chemical or biological environment. We employ here large‐scale quantum chemical calculations to study the light‐capturing properties of retinal in recently designed human cellular retinol binding protein II (hCRBPII) variants (Wang et al. Science, 2012 , 338, 1340–1343). Our calculations show that these proteins absorb across a large part of the visible spectrum by combined polarization and electrostatic effects. These effects stabilize the ground or excited state energy levels of the retinal by perturbing the Schiff‐base or β‐ionone moieties of the chromophore, which in turn modulates the amount of charge transfer within the molecule. Based on the predicted tuning principles, we design putative in silico mutations that further shift the absorption properties of retinal in hCRBPII towards the ultraviolet and infrared regions of the spectrum.     

Absorption of schiff-base retinal chromophores in vacuo

The absorption spectrum of the all-trans retinal chromophore in the protonated Schiff-base form, that is, the biologically relevant form, has been measured in vacuo, and a maximum is found at 610 nm. The absorption of retinal proteins has hitherto been compared to that of protonated retinal in methanol, where the absorption maximum is at 440 nm. In contrast, the new gas-phase absorption data constitute a well-defined reference for spectral tuning in rhodopsins in an environment devoid of charges and dipoles. They replace the misleading comparison with absorption properties in solvents and lay the basis for reconsidering the molecular mechanisms of color tuning in the large family of retinal proteins. Indeed, our measurement directly shows that protein environments in rhodopsins are blue- rather than red shifting the absorption. The absorption of a retinal model chromophore with a neutral Schiff base is also studied. The data explain the significant blue shift that occurs when metharhodopsin I becomes deprotonated as well as the purple-to-blue transition of bacteriorhodopsin upon acidification.     

Protein elasticity determined by pressure tuning of the tyrosine residue of ubiquitin

We determined the isotropic, isothermal compressibility of ubiquitin by pressure tuning spectral holes burnt into the red edge of the absorption spectrum of the single tyrosine residue. The pressure shift is perfectly linear with burn frequency. From these data, a compressibility of 0.086 GPa(-1) in the local environment of the tyrosine residue could be determined. This value fits nicely into the range known for proteins. Although the elastic behavior at low temperatures does not show any unusual features, the pressure tuning behavior at room temperature is quite surprising: the pressure-induced spectral shift is close to zero, even up to very high pressure levels of 0.88 GPa, well beyond the denaturation point. The reason for this behavior is attributed to equally strong blue as well as red spectral pressure shifts resulting in an average pressure-induced solvent shift that is close to zero.     

The Effective Conjugation Length Is Responsible for the Red/Green Spectral Tuning in the Cyanobacteriochrome Slr1393g3

The origin of the spectral shift from a red‐ to a green‐absorbing form in a cyanobacteriochrome, Slr1393g3, was identified by combined quantum mechanics/molecular mechanics simulations. This protein, related to classical phytochromes, carries the open‐chain tetrapyrrole chromophore phycocyanobilin. Our calculations reveal that the effective conjugation length in the chromophore becomes shorter upon conversion from the red to the green form. This is related to the planarity of the entire chromophore. A large distortion was found for the terminal pyrrole rings A and D; however, the D ring contributes more strongly to the photoproduct tuning, despite a larger change in the twist of the A ring. Our findings implicate that the D ring twist can be exploited to regulate the absorption of the photoproduct. Hence, mutations that affect the D ring twist can lead to rational tuning of the photoproduct absorption, allowing the tailoring of cyanobacteriochromes for biotechnological applications such as optogenetics and bioimaging.     

Deciphering the Spectral Tuning Mechanism in Proteorhodopsin: The Dominant Role of Electrostatics Instead of Chromophore Geometry

Proteorhodopsin (PR) is a photoactive proton pump found in marine bacteria. There are two phenotypes of PR exhibiting an environmental adaptation to the ocean's depth which tunes their maximum absorption: blue-absorbing proteorhodopsin (BPR) and green-absorbing proteorhodopsin (GPR). This blue/green color-shift is controlled by a glutamine to leucine substitution at position 105 which accounts for a 20 nm shift. Typically, spectral tuning in rhodopsins is rationalized by the external point charge model but the Q105L mutation is charge neutral. To study this tuning mechanism, we employed the hybrid QM/MM method with sampling from molecular dynamics. Our results reveal that the positive partial charge of glutamine near the C₁₄−C₁₅ bond of retinal shortens the effective conjugation length of the chromophore compared to the leucine residue. The derived mechanism can be applied to explain the color regulation in other retinal proteins and can serve as a guideline for rational design of spectral shifts.     

Accurate evaluation of the absorption maxima of retinal proteins based on a hybrid QM/MM method

Here we improved our hybrid QM/MM methodology (Houjou et al. J Phys Chem B 2001, 105, 867) for evaluating the absorption maxima of photoreceptor proteins. The renewed method was applied to evaluation of the absorption maxima of several retinal proteins and photoactive yellow protein. The calculated absorption maxima were in good agreement with the corresponding experimental data with a computational error of <10 nm. In addition, our calculations reproduced the experimental gas-phase absorption maxima of model chromophores (protonated all-trans retinal Schiff base and deprotonated thiophenyl-p-coumarate) with the same accuracy. It is expected that our methodology allows for definitive interpretation of the spectral tuning mechanism of retinal proteins.     

Spectral characterization of chlorophyll fluorescence in barley leaves during linear heating. Analysis of high-temperature fluorescence rise around 60 degrees C

The spectral characteristics of chlorophyll fluorescence and absorption during linear heating of barley leaves within the range 25-75 degreesC (fluorescence temperature curve, FTC) were studied. Leaves with various content of light harvesting complexes (green, Chl b-less chlorina f2 and intermittent light grown) revealing different types of FTC were used. Differential absorption, emission and excitation spectra documented four characteristic phases of the FTC. The initial two FTC phases (a rise in the 46-49 degreesC region and a subsequent decrease to about 55 degreesC) mostly reflected changes in the fluorescence quantum yield peaking at about 685 nm. A steep second fluorescence rise at 55-61 degreesC was found to originate from a short-wavelength Chl a spectral form (emission maximum at 675 nm) causing a gradual blue shift of the emission spectra. In this temperature range, a clear correspondence of the blue shift in the emission and absorption spectra was found. We suggest that the second fluorescence rise in FTC reflects a weakening of the Chl a-protein interaction in the thylakoid membrane.     

NMR Backbone Assignment of Large Proteins by Using 13Cα‐Only Triple‐Resonance Experiments

Nuclear magnetic resonance (NMR) is a powerful tool to interrogate protein structure and dynamics residue by residue. However, the prerequisite chemical‐shift assignment remains a bottleneck for large proteins due to the fast relaxation and the frequency degeneracy of the ¹³C_α nuclei. Herein, we present a covariance NMR strategy to assign the backbone chemical shifts by using only HN(CO)CA and HNCA spectra that has a high sensitivity even for large proteins. By using the peak linear correlation coefficient (LCC), which is a sensitive probe even for tiny chemical‐shift displacements, we correctly identify the fidelity of approximately 92 % cross‐peaks in the covariance spectrum, which is thus a significant improvement on the approach developed by Snyder and Brüschweiler (66 %) and the use of spectral derivatives (50 %). Thus, we calculate the 4D covariance spectrum from HN(CO)CA and HNCA experiments, in which cross‐peaks with LCCs above a universal threshold are considered as true correlations. This 4D covariance spectrum enables the sequential assignment of a 42 kDa maltose binding protein (MBP), in which about 95 % residues are successfully assigned with a high accuracy of 98 %. Our LCC approach, therefore, paves the way for a residue‐by‐residue study of the backbone structure and dynamics of large proteins.     

Effects of Substituents on Synthetic Analogs of Chlorophylls. Part 4: How Formyl Group Location Dictates the Spectral Properties of Chlorophylls b,d and f

Photosynthetic organisms are adapted to light characteristics in their habitat in part via the spectral characteristics of the associated chlorophyll pigments, which differ in the position of a formyl group around the chlorin macrocycle (chlorophylls b, d, f) or no formyl group (chlorophyll a). To probe the origin of this spectral tuning, the photophysical and electronic structural properties of a new set of synthetic chlorins are reported. The zinc and free base chlorins have a formyl group at either the 2‐ or 3‐position. The four compounds have fluorescence yields in the range 0.19–0.28 and singlet excited‐state lifetimes of ca 4 ns for zinc chelates and ca 8 ns for the free base forms. The photophysical properties of the 2‐ and 3‐formyl zinc chlorins are similar to those observed previously for 13‐formyl or 3,13‐diformyl chlorins, but differ markedly from those for 7‐formyl analogs. Molecular‐orbital characteristics obtained from density functional theory (DFT) calculations were used as input to spectral simulations employing the four‐orbital model. The analysis has uncovered the key changes in electronic structure engendered by the presence/location of a formyl group at various macrocycle positions, which is relevant to understanding the distinct spectral properties of the natural chlorophylls a, b, d and f.     

Pigment-pigment and pigment-protein interactions in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower

Plants contain water-soluble chlorophyll-binding proteins (WSCPs) that function neither as antennas nor as components of light-induced electron transfer of photosynthesis but are likely constituents of regulatory protective pathways in particular under stress conditions. This study presents results on the spectroscopic properties of recombinant WSCP from cauliflower reconstituted with chlorophyll b (Chl b) alone or with mixtures of Chl a and Chl b. Two types of experiments were performed: (a) measurements of stationary absorption spectra at 77 and 298 K and CD spectra at 298 K and (b) monitoring of laser flash-induced transient absorption changes with a resolution of 200 fs in the time domain of up to 100 ps. On the basis of a theoretical analysis outlined by Renger et al. (J. Phys. Chem. B 2007, 111, 10487) the data obtained in part (a) are interpreted within a model where tetrameric WSCP binds predominantly two Chl molecules in the form of an excitonically coupled "open sandwich" dimer with a tilt angle of about 30 degrees between the chlorin planes. The time-resolved measurements on Chl a/Chl b heterodimers are described by two exponential kinetics with time constants of 400 fs and 7 ps. These kinetics are assumed to reflect a heterogeneous population of WSCPs with Chl dimers either in excitonic coupled "open sandwich" or weakly coupled geometric arrays. The 400 fs component is assigned to excited-state relaxations from the upper to the lower excitonic level of the strongly coupled "open sandwich" dimer, while the 7-8 ps component probably indicates excitation energy transfer from 1Chl b* to Chl a in a dimer array with weak coupling due to significantly longer mutual distances between the chlorin rings.     

Effect of Explicit Water Molecules on the Color‐Tuning Mechanism of the Firefly

The contributions of explicit water molecules to color‐tuning mechanism of firefly were studied. The explicit water molecules cause two different structures in the geometrical parameters of keto(‐1) both in vacuo and aqueous solution. There are somewhat larger influences on absorption and emission spectra. When water molecules were added only on the side of benzothiazole ring, the spectra shift to the blue. In contrast, when waters were added only on the side of the thiazoline ring, the spectra shift to red. In a word, the color modulation of the emitted light depends on charge redistribution of molecule keto(‐1), mainly the charge change of the benzothiazole and thiazole rings at the two terminal in keto(‐1).     

Construction of Chlorosomal Rod Self‐Aggregates in the Solid State on Any Substrates from Synthetic Chlorophyll Derivatives Possessing an Oligomethylene Chain at the 17‐Propionate Residue

Chlorosomes are one of the most unique natural light‐harvesting antennas and their supramolecular nanostructures are still under debate. Chlorosomes contain bacteriochlorophyll (BChl)‐c, d and e molecules and these pigments self‐aggregate under a hydrophobic environment inside a chlorosome. The self‐aggregates are mainly constructed by the following three interactions: hydrogen bonding, coordination bonding and π–π stacking. Supramolecular nanostructures of self‐aggregated BChls have been widely investigated by spectroscopic and microscopic techniques. Model compounds of such chlorosomal BChl molecules have been synthesized and the effects of esterified long alkyl chains at the 17‐propionate residue for their self‐aggregation have been studied. Structurally simple zinc chlorophyll derivatives possessing an oligomethylene chain as the esterifying group at the 17‐propionate residue were prepared as chlorosomal BChl models. The synthetic zinc BChls self‐aggregated in nonpolar organic solvents to give precipitates. The resulting insoluble self‐aggregated solids were investigated on a variety of substrates, including hydrophobic, neutral and hydrophilic substrates, by visible absorption, circular dichroism and polarized light absorption spectroscopies, as well as atomic force, transmission electron and scanning electron microscopies. The self‐aggregates of synthetic Zn‐BChls formed rods with an approximately 5 nm diameter and wires with further elongated growth of the rods (aspect ratio >200). The diameter size was consistent with that estimated for natural chlorosomal rods in a filamentous anoxygenic phototroph, Chloroflexus aurantiacus. The supramolecular formation and stability of the rod on the examined substrates depended on the length of an oligomethylene chain at the 17‐propionate residue as well as on the surface properties. Especially, the number of the 5 nm rods on the substrates increased with an elongation of the chain.     

水/醇溶共轭聚合物界面材料及其在光电器件中的应用

相对于传统的无机半导体器件,以有机半导体(特别是聚合物半导体)材料为基础的有机光电器件,可采用与传统印刷技术(例如喷墨打印、卷对卷印刷等)相结合的溶液加工方式制备低成本、大面积、柔性光电器件,因而成为广泛关注的焦点,并得到了快速发展.实现溶液加工的高效有机光电器件的一个关键问题是界面问题——如何避免溶液加工时有机层间的互溶以及如何实现可印刷稳定金属电极的高效电子注入等.水/醇溶性共轭聚合物的迅速发展为解决溶液加工多层有机光电器件所面临的界面问题提供了有效手段.研究发现,水/醇溶共轭聚合物不但可以有效避免溶液加工多层器件中的界面互溶,而且还可与高功函数的稳定金属发生界面偶极相互作用而增强其电子注入,从而解决了高功函数稳定金属电子注入的难题,为实现全溶液加工的高效印刷有机光电器件提供了可行的方案.本文介绍了近年来本课题组在水/醇溶共轭聚合物阴极界面材料及器件应用方面的研究进展,并对水/醇溶共轭聚合物阴极界面材料在聚合物发光二极管和聚合物太阳电池中的工作机理进行了探讨.     

Far-infrared spectroscopy on free-standing protein films under defined temperature and hydration control

The functionality of proteins is governed by their dynamics. We have performed a systematic investigation on four different proteins in the far-infrared spectral region under control of the two external parameters that have the strongest influence on the dynamics, namely temperature and hydration. The absorption measurements covering the frequency range from 40 cm(-1) to 690 cm(-1) (1-20 THz) close the gap between the well-studied mid-infrared and the recent THz investigations. By preparing the proteins as free-standing films, we achieve unprecedented reproducibility. Besides a featureless slope in the THz range, we can identify absorption peaks characteristic for each protein and others common to several proteins. We fit the spectra to extract the peak positions and suggest assignments for them. The far-infrared absorption spectra of all proteins are basically independent on hydration. By a detailed analysis of the sorption isotherms this can be explained by the low absorption of biological water, which resembles more the behavior of ice than that of liquid water.     

Opsin Expression in Human Epidermal Skin

Human skin is constantly exposed to solar light containing visible and ultraviolet radiation (UVR), a powerful skin carcinogen. UVR elicits cellular responses in epidermal cells via several mechanisms: direct absorption of short‐wavelength UVR photons by DNA, oxidative damage caused by long‐wavelength UVR, and, as we recently demonstrated, via a retinal‐dependent G protein‐coupled signaling pathway. Because the human epidermis is exposed to a wide range of light wavelengths, we investigated whether opsins, light‐activated receptors that mediate photoreception in the eye, are expressed in epidermal skin to potentially serve as photosensors. Here we show that four opsins—OPN1‐SW, OPN2, OPN3 and OPN5—are expressed in the two major human epidermal cell types, melanocytes and keratinocytes, and the mRNA expression profile of these opsins does not change in response to physiological UVR doses. We detected two OPN3 splice variants present in similar amounts in both cell types and three OPN5 splice isoforms, two of which encode truncated proteins. Notably, OPN2 and OPN3 mRNA were significantly more abundant than other opsins and encoded full‐length proteins. Our results demonstrate that opsins are expressed in epidermal skin cells and suggest that they might initiate light–induced signaling pathways, possibly contributing to UVR phototransduction.