Water-soluble Chlorophyll-binding Protein Research Articles

Theory of 2D electronic spectroscopy of water soluble chlorophyll-binding protein (WSCP): Signatures of Chl b derivate.

We investigate how electronic excitations and subsequent dissipative dynamics in the water soluble chlorophyll-binding protein (WSCP) are connected to features in two-dimensional (2D) electronic spectra, thereby comparing results from our theoretical approach with experimental data from the literature. Our calculations rely on third-order response functions, which we derived from a second-order cumulant expansion of the dissipative dynamics involving the partial ordering prescription, assuming a fast vibrational relaxation in the potential energy surfaces of excitons. Depending on whether the WSCP complex containing a tetrameric arrangement of pigments composed of two dimers with weak excitonic coupling between them binds the chlorophyll variant Chl a or Chl b, the resulting linear absorption and circular dichroism spectra and particularly the 2D spectra exhibit substantial differences in line shapes. These differences between Chl a WSCP and Chl b WSCP cannot be explained by the slightly modified excitonic couplings within the two variants. In the case of Chl a WSCP, the assumption of equivalent dimer subunits facilitates a reproduction of substantial features from the experiment by the calculations. In contrast, for Chl b WSCP, we have to assume that the sample, in addition to Chl b dimers, contains a small but distinct fraction of chemically modified Chl b pigments. The existence of such Chl b derivates has been proposed by Pieper et al. [J. Phys. Chem. B 115, 4042 (2011)] based on low-temperature absorption and hole-burning spectroscopy. Here, we provide independent evidence.

Identification of Residues Potentially Involved in Optical Shifts in the Water-Soluble Chlorophyll a-Binding Protein through Molecular Dynamics Simulations.

Reversible light and thermally induced spectral shifts are universally observed in a wide variety of pigment-protein complexes at temperatures ranging from cryogenic to ambient. In this paper, we employed large-scale molecular dynamics (MD) simulations of a prototypical pigment-protein complex to better understand these shifts at a molecular scale. Although multiple mechanisms have been proposed over the years, no verification of these proposals via MD simulations has thus far been performed; our work represents the first step in this direction. From simulations of the water-soluble chlorophyll-binding protein complex, we determined that rearrangements of long hydrogen bonds were unlikely to be the origin of the multiwell landscape features necessary to explain observed spectral shifts. We also assessed small motions of amino acid residues and identified side chain rotations of some of these residues as likely candidates for the origin of relevant multiwell landscape features. The protein free-energy landscapes associated with side chain rotations feature energy barriers of around 1100-1600 cm-1, in agreement with optical spectroscopy results, with the most promising residue type associated with experimental signatures being serine, which possesses a symmetric triple-well landscape and moment of inertia of a relevant magnitude.

Exciton interactions of chlorophyll tetramer in water-soluble chlorophyll-binding protein BoWSCP

The exciton interaction of four chlorophyll a (Chl a) molecules in a symmetrical tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP was analyzed in the pH range of 3–11. Exciton splitting ΔE = 232 ± 2 cm−1 of the Qy band of Chl a into two subcomponents with relative intensities of 78.1 ± 0.7 % and 21.9 ± 0.7 % was determined by a joint decomposition of the absorption and circular dichroism spectra into Gaussian functions. The exciton coupling parameters were calculated based on the BoWSCP atomic structure in three approximations: the point dipole model, the distributed atomic monopoles, and direct ab initio calculations in the TDDFT/PCM approximation. The Coulomb interactions of monomers were calculated within the continuum model using three values of optical permittivity. The models based on the properties of free Chl a in solution suffer from significant errors both in estimating the absolute value of the exciton interaction and in the relative intensity of exciton transitions. Calculations within the TDDFT/PCM approximation reproduce the experimentally determined parameters of the exciton splitting and the relative intensities of the exciton bands. The following factors of pigment-protein and pigment–pigment interactions were examined: deviation of the macrocycle geometry from the planar conformation of free Chl; the formation of hydrogen bonds between the macrocycle and water molecules; the overlap of wave functions of monomers at close distances. The most significant factor is the geometrical deformation of the porphyrin macrocycle, which leads to an increase in the dipole moment of Chl monomer from 5.5 to 6.9 D and to a rotation of the dipole moment by 15° towards the cyclopentane ring. The contributions of resonant charge-transfer states to the wave functions of the Chl dimer were determined and the transition dipole moments of the symmetric and antisymmetric charge-transfer states were estimated.

Photochemical reactions of chlorophyll tetramer in water-soluble chlorophyll-binding protein BoWSCP

The absorption dynamics of chlorophyll a in a symmetric tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP was measured by the broadband femtosecond laser pump-probe spectroscopy within the range from 400 to 780 nm with a time resolution of 20 f-200 ps. When BoWSCP was excited in the region of the Soret band at a wavelength of 430 nm, a nonradiative intramolecular conversion S3 → S1 was observed with a characteristic time of 83 ± 9 fs. When the complex was excited in the region of the Qy band at a wavelength of 670 nm, a relaxation transition between two excitonic states of the chlorophyll dimer was observed with a time of 105 ± 10 fs. Absorption spectra of excited singlet states S1 and S3 of chlorophyll a were obtained. It has been demonstrated that the delocalization of the excited state between exciton-coupled Chl molecules in the BoWSCP tetramer changes in time and depends on the excitation energy. Upon excitation of BoWSCP, an ultrafast photochemical reaction is observed in the Soret region, apparently due to the reduction of tryptophan in the vicinity of chlorophyll.

Femtosecond Dynamics of Excited States of Chlorophyll Tetramer in Water-Soluble Chlorophyll-Binding Protein BoWSCP.

The paper reports on the absorption dynamics of chlorophyll a in a symmetric tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP. It was measured by a broadband femtosecond laser pump-probe spectroscopy within the range from 400 to 750 nm and with a time resolution of 20 fs-200 ps. When BoWSCP was excited in the region of the Soret band at a wavelength of 430 nm, nonradiative intramolecular conversion S3→S1 was observed with a characteristic time of 83 ± 9 fs. When the complex was excited in the region of the Qy band at 670 nm, relaxation transition between two excitonic states of the chlorophyll dimer was observed in the range of 105 ± 10 fs. Absorption spectra of the excited singlet states S1 and S3 of chlorophyll a were obtained. The delocalization of the excited state between exciton-coupled Chl molecules in BoWSCP tetramer changed in time and depended on the excitation energy. When BoWSCP is excited in the Soret band region, an ultrafast photochemical reaction is observed. This could result from the reduction of tryptophan in the vicinity of chlorophyll.

Signatures of intramolecular vibrational and vibronic Q_{mathrm{x}}–Q_{mathrm{y}} coupling effects in absorption and CD spectra of chlorophyll dimers

An electron-vibrational coupling model that includes the vibronic (non-adiabatic) coupling between the Q_{mathrm{y}} and Q_{mathrm{x}} transitions of chlorophyll (Chl), created by Reimers and coworkers (Scientific Rep. 3, 2761, 2013) is extended here to chlorophyll dimers with interchlorophyll excitonic coupling. The model is applied to a Chl a dimer of the water-soluble chlorophyll binding protein (WSCP). As for isolated chlorophyll, the vibronic coupling is found to have a strong influence on the high-frequency vibrational sideband in the absorption spectrum, giving rise to a band splitting. In contrast, in the CD spectrum the interplay of vibronic coupling and static disorder leads to a strong suppression of the vibrational sideband in excellent agreement with the experimental data. The conservative nature of the CD spectrum in the low-energy region is found to be caused by a delicate balance of the intermonomer excitonic coupling between the purely electronic Q_{mathrm{y}} transition and the Q_{mathrm{y}} transition involving intramolecular vibrational excitations on one hand and the coupling to higher-energy electronic transitions on the other hand.

Exact simulation of pigment-protein complexes unveils vibronic renormalization of electronic parameters in ultrafast spectroscopy

The primary steps of photosynthesis rely on the generation, transport, and trapping of excitons in pigment-protein complexes (PPCs). Generically, PPCs possess highly structured vibrational spectra, combining many discrete intra-pigment modes and a quasi-continuous of protein modes, with vibrational and electronic couplings of comparable strength. The intricacy of the resulting vibronic dynamics poses significant challenges in establishing a quantitative connection between spectroscopic data and underlying microscopic models. Here we show how to address this challenge using numerically exact simulation methods by considering two model systems, namely the water-soluble chlorophyll-binding protein of cauliflower and the special pair of bacterial reaction centers. We demonstrate that the inclusion of the full multi-mode vibronic dynamics in numerical calculations of linear spectra leads to systematic and quantitatively significant corrections to electronic parameter estimation. These multi-mode vibronic effects are shown to be relevant in the longstanding discussion regarding the origin of long-lived oscillations in multidimensional nonlinear spectra.

Direct Assembly in Aqueous Solutions of Stable Chlorophyllide Complexes with Type II Water-soluble Chlorophyll Proteins.

Water-soluble chlorophyll-binding proteins (WSCPs) from Brassicaceae constitute a small family of non-photosynthetic proteins that may provide a useful benchmark and model system for studying molecular aspects of chlorophyll-protein interactions such as the tuning of absorption and emission spectra, and binding selectivity. WSCP apo-proteins are readily expressed by recombinant DNA techniques and can be assembled in vitro with natural and synthetic chlorophyll derivatives. The complexes with native chlorophylls are exceptionally stable toward thermal dissociation and protein denaturation due to hydrophobic interactions with the chlorophyll's phytyl chains that stabilize the core of the WSCP tetrameric complexes. However, assembly requires the use of detergents or water-in-oil emulsions to introduce the hydrophobic pigments into the water-soluble apo-proteins. Here, we explore the direct assembly of recombinant WSCPs with the water-soluble phytyl-free chlorophyll analogue chlorophyllide a in aqueous solutions. We show that the complexes formed by mixing chlorophyllide and WSCP apo-proteins are exclusively tetrameric, and while they lack the extreme thermostability of the respective chlorophyll complexes, they are still thermostable up to around 60°C. Their absorption and CD spectra are very similar to the chlorophyll complexes albeit slight peak shifts and broadening of the bands indicate variations in pigment and protein conformations, and less rigid structures. Simplifying the assembly process of WSCPs opens new possibilities for their use in modelling natural chlorophyll-protein complexes, and as templates for designing novel artificial protein-pigment complexes.

ИССЛЕДОВАНИЕ ФОТОХИМИЧЕСКИХ СВОЙСТВ И ФИЛОГЕНИИ РАСТИТЕЛЬНЫХ БЕЛКОВ СЕМЕЙСТВА WSCP

Here we show the ability of the chlorophyll associated with proteins of the WSCP family (Water-Soluble Chlorophyll-binding Proteins) to photosensitize oxidative redox reactions. Irradiation with red light of the recombinant chlorophyll-protein complexes WSCP subclasses IIa and IIb in the presence of an electron donor (NADH) led to oxidation of the donor, i.e., these pigment-protein complexes showed photochemical activity. Meanwhile there was no photodestruction of chlorophyll associated with WSCP, which indicates the photocatalytic nature of the detected redox reaction. The kinetic constants of NADH photooxidation were higher for WSCP subclass IIa (BoWSCP) than for subclass IIb (LvWSCP). To explain the difference in the photosensitizing activity of representatives of different WSCP subclasses, bioinformatic analysis of class II proteins of this family was carried out. For this purpose, we searched for members of the WSCP family in the UniProt protein sequence database using the BLAST search algorithm, followed by their multiple alignment and construction of a phylogenetic tree using the EMBL-EBI Clustal Omega web service and the MEGA7 program. Bioinformatic analysis has confirmed the phylogenetic division of the WSCP class II protein family into two subclasses, previously established on the basis of the difference in their physicochemical properties. It was suggested that phylogeny is related to the photochemical activity of representatives of different subclasses in the WSCP family.

Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase

BackgroundLytic polysaccharide monooxygenases (LPMOs) are indispensable redox enzymes used in industry for the saccharification of plant biomass. LPMO-driven cellulose oxidation can be enhanced considerably through photobiocatalysis using chlorophyll derivatives and light. Water soluble chlorophyll binding proteins (WSCPs) make it is possible to stabilize and solubilize chlorophyll in aqueous solution, allowing for in vitro studies on photostability and ROS production. Here we aim to apply WSCP–Chl a as a photosensitizing complex for photobiocatalysis with the LPMO, TtAA9.ResultsWe have in this study demonstrated how WSCP reconstituted with chlorophyll a (WSCP–Chl a) can create a stable photosensitizing complex which produces controlled amounts of H2O2 in the presence of ascorbic acid and light. WSCP–Chl a is highly reactive and allows for tightly controlled formation of H2O2 by regulating light intensity. TtAA9 together with WSCP–Chl a shows increased cellulose oxidation under low light conditions, and the WSCP–Chl a complex remains stable after 24 h of light exposure. Additionally, the WSCP–Chl a complex demonstrates stability over a range of temperatures and pH conditions relevant for enzyme activity in industrial settings.ConclusionWith WSCP–Chl a as the photosensitizer, the need to replenish Chl is greatly reduced, enhancing the catalytic lifetime of light-driven LPMOs and increasing the efficiency of cellulose depolymerization. WSCP–Chl a allows for stable photobiocatalysis providing a sustainable solution for biomass processing.

On the Stability of the Water-Soluble Chlorophyll-Binding Protein (WSCP) Studied by Molecular Dynamics Simulations.

The water-soluble chlorophyll-binding protein (WSCP) is assumed to be not a part of the photosynthetic process. Applying molecular dynamics (MD) simulations, we aimed to obtain insight into the exceptional stability of WSCP. We analyzed dynamical features such as the hydrogen bond network, flexibility, and force distributions. The WSCP structure contains two cysteines at the interfaces of every protein chain, which are in close contact with the cysteines of the other dimer. We tested if a connection of these cysteines between different protein chains influences the dynamical behavior to investigate any influences on the thermal stability. We find that the hydrogen bond network is very stable regardless of the presence or absence of the hypothetical disulfide bridges and/or the chlorophyll units. Furthermore, it is found that the phytyl chains of the chlorophyll units are extremely flexible, much more than what is seen in crystal structures. Nonetheless, they seem to protect a photochemically active site of the chlorophylls over the complete simulation time. Finally, we also find that a cavity in the chlorophyll-surrounding sheath exists, which may allow access for individual small molecules to the core of WSCP.

Water Soluble Chlorophyll-Binding Proteins of Plants: Structure, Properties and Functions

Water Soluble Chlorophyll-Binding Proteins of Plants: Structure, Properties and Functions

How Well Does the Hole-Burning Action Spectrum Represent the Site-Distribution Function of the Lowest-Energy State in Photosynthetic Pigment-Protein Complexes?

For the first time, we combined Monte Carlo and nonphotochemical hole burning (NPHB) master equation approaches to allow for ultrahigh-resolution (<0.005 cm-1, smaller than the typical homogeneous line widths at 5 K) simulations of the NPHB spectra of dimers and trimers of interacting pigments. These simulations reveal significant differences between the zero-phonon hole (ZPH) action spectrum and the site-distribution function (SDF) of the lowest-energy state. The NPHB of the lowest-energy pigment, following the excitation energy transfer (EET) from the higher-energy pigments which are excited directly, results in the shifts of all excited states. These shifts affect the ZPH action spectra and EET times derived from the widths of the spectral holes burned in the donor-dominated regions. The effect is present for a broad variety of realistic antihole functions, and it is maximal at relatively low values of interpigment coupling (V ≤ 5 cm-1) where the use of the Förster approximation is justified. These findings need to be considered in interpreting various optical spectra of photosynthetic pigment-protein complexes for which SDFs (describing the inhomogeneous broadening) are often obtained directly from the ZPH action spectra. Water-soluble chlorophyll-binding protein (WSCP) was considered as an example.

Water Soluble Chlorophyll-Binding Proteins of Plants: Structure, Properties and Functions

Water soluble chlorophyll-binding proteins (WSCPs) of higher plants differ from most proteins containing chlorophyll orbacteriochlorophyll in that they are soluble in watr and are neither embedded in the lipid membrane nor directly involved in the process of photosynthesis. Chlorophyll molecules in WSCPs ensembles are packed in dimers within the hydrophobic zone of the protein matrix, similar to the structure of a chlorophyll "special pair" in the reaction centers of phototrophs. This fact together with the detected photosensitizing activity of WSCPs makes it possible to consider these proteins as a promising object for modelling the evolutionary prototypes of the photosynthetic apparatus, as well as for developing the artificial solar energy converters. There are two classes of proteins in the WSCP family, class I and class II the representatives of these classes have a weak degree of homology in the primary structure, but a high degree of similarity in the tertiary and quaternary structure. One of the features of class I WSCPs is photoconversion, that is, to change the structure and spectral properties of the chromophore under the action of light. The functions of WSCPs in the plant are thought to be associated with stress protection.

Wavelength-Dependent Exciton-Vibrational Coupling in the Water-Soluble Chlorophyll Binding Protein Revealed by Multilevel Theory of Difference Fluorescence Line-Narrowing.

One of the most powerful line-narrowing techniques used to unravel the homogeneous lineshapes of inhomogeneously broadened systems is difference fluorescence line-narrowing spectroscopy. When this spectroscopy was applied to multichromophoric systems so far, the spectra were analyzed by an effective two-level system approach, composed of the electronic ground state and the lowest exciton state. An effective Huang-Rhys factor was assigned for the coupling of this state to the vibrations. Here, we extend this approach by including a multilevel line shape theory, which takes into account the excitonic coupling between pigments and thereby the effect of the delocalization of the excited states explicitly. In this way, it becomes possible to extract the spectral density of the local exciton-vibrational coupling. The theory is applied to the recombinant water-soluble chlorophyll binding protein reconstituted with chlorophyll a or b and reveals a significant decrease of the Huang-Rhys factor of the local exciton-vibrational coupling with decreasing transition energy of the chlorophylls. This decrease could be due to the increase in steric interactions reducing the flexibility of the environment and red-shifting the site energy of the pigments.

Vibrational damping effects on electronic energy relaxation in molecular aggregates

Representation of molecular vibrational degrees of freedom by independent harmonic oscillators, when describing electronic spectra or electronic excitation energy transport, raises unfavourable effects as vibrational energy relaxation becomes inaccessible. A standard theoretical description is extended in this paper by including both electronic-phonon and vibrational-phonon couplings. Using this approach we have simulated a model pigment-protein system and have shown that intermode coupling leads to the quenching of pigment vibrational modes, and to the redistribution of fluctuation spectral density with respect to the electronic excitations. Moreover, new energy relaxation pathways, opened by the vibrational-phonon interaction, allow to reach the electronic excited state equilibrium quicker in the naturally occurring water soluble chlorophyll binding protein (WSCP) aggregate, demonstrating the significance that the damping of molecular vibrations has for the rate of the intramolecular energy relaxation process.

An unusual role for the phytyl chains in the photoprotection of the chlorophylls bound to Water-Soluble Chlorophyll-binding Proteins

Water-Soluble Chlorophyll Proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins which tetramerize upon binding four chlorophyll (Chl) molecules. The bound Chls are highly photostable, despite the lack of bound carotenoids known, in Chl-containing photosynthetic proteins, to act as singlet oxygen and Chl triplet (3Chl) quenchers. Although the physiological function of WSCPs is still unclear, it is likely to be related to their biochemical stability and their resistance to photodegradation. To get insight into the origin of this photostability, the properties of the 3Chl generated in WSCPs upon illumination were investigated. We found that, unlike the excited singlet states, which are excitonic states, the triplet state is localized on a single Chl molecule. Moreover, the lifetime of the 3Chl generated in WSCPs is comparable to that observed in other Chl-containing systems and is reduced in presence of oxygen. In contrast to previous observations, we found that WSCP actually photosensitizes singlet oxygen with an efficiency comparable to that of Chl in organic solvent. We demonstrated that the observed resistance to photooxidation depends on the conformation of the phytyl moieties, which in WSCP are interposed between the rings of Chl dimers, hindering the access of singlet oxygen to the oxidizable sites of the pigments.

Plants water soluble chlorophyll binding proteins act as enzyme-inhibitor pair

The hydrophilic water-soluble chlorophyll binding proteins (WSCP) which form complex with chlorophyll molecules have been numerously isolated from the chloroplasts of plants. Although, their molecular properties have been partly characterized, but their physio-biochemical roles are still unclear in the photosynthesizing organs. In this study, using bioinformatic tools WSCP pair were predicted to act as hydrolase and hydrolase inhibitor towards chlorophyll molecules. To enhance our information regarding the possible functions of WSCP, we cloned WSCP1 and WSCP2 cDNAs from Chenopodium album L. and Brassica oleracea L. leaves and expressed them as soluble maltose-binding fusion proteins in Escherichia coli. The purified fused products were subjected to chlorophyll hydrolyzing activity in vitro. The results showed that WSCP1 and WCSP2 are antagonistically involved in chlorophyll breakdown, while WSCP1 acts as chlorophyll hydrolyzing enzyme (with the hydrolysis rate of about 40% per 12 h), WSCP2 exerts inhibitory activity (with the inhibition rate of about 38% per 12 h) towards chlorophyll hydrolysis. This is the first ever time report speculates the hydrolase/inhibitory roles for WSCP and proposes that the relative activity of WSCP pair might balance and regulate the chlorophyll breakdown process in the photosynthetic apparatus of plants. It may open the new gate to investigate the potent roles of WSCP in plant system.

Chlorophyll Binding Ability of Non-chloroplastic DUF538 Protein Superfamily in Plants

The type-1 water soluble chlorophyll binding proteins (WSCP1) have been generally known as chlorophyll extractors and transporters from the thylakoid membrane to the chloroplast envelope, where the membrane bound chlorophyllase catabolizes the chlorophyll. Despite the type-2 WSCP, WSCP1 has been known to be located in the chloroplasts of the green plants. In the present study, the non-chloroplastic protein superfamily containing domain of unknown function 538 (DUF538) as functional homologue of type-1 WSCP has been identified in plants. The structural similarities/differences and the cellular locations of Celosia cristata DUF538 and Chenopodium album WSCP1 were predicted by using computational tools and the chlorophyll binding abilities of their purified maltose binding protein-fused forms were estimated by maltose binding affinity method. It was predicted that despite CaWSCP1, CcDUF538 is a non-chloroplastic protein. The chlorophyll binding abilities of the recombinant fusion forms of test WSCP1 and DUF538 were estimated to be about 58 and 56%, respectively. Considering DUF538 as stress-induced protein, it was speculated that they may form complex with chlorophyll molecules or their hydrolyzed products out of chloroplasts to proceed the chlorophyll breakdown and nitrogen/carbon recycling in stress-challenged plants.

Fine Tuning of Chlorophyll Spectra by Protein-Induced Ring Deformation.

The ability to tune the light-absorption properties of chlorophylls by their protein environment is the key to the robustness and high efficiency of photosynthetic light-harvesting proteins. Unfortunately, the intricacy of the natural complexes makes it very difficult to identify and isolate specific protein-pigment interactions that underlie the spectral-tuning mechanisms. Herein we identify and demonstrate the tuning mechanism of chlorophyll spectra in type II water-soluble chlorophyll binding proteins from Brassicaceae (WSCPs). By comparing the molecular structures of two natural WSCPs we correlate a shift in the chlorophyll red absorption band with deformation of its tetrapyrrole macrocycle that is induced by changing the position of a nearby tryptophan residue. We show by a set of reciprocal point mutations that this change accounts for up to 2/3 of the observed spectral shift between the two natural variants.