Water-soluble Chlorophyll Protein Research Articles

Controlling Vibronic Coupling in Chlorophyll Proteins: The Effects of Excitonic Delocalization and Vibrational Localization.

Vibrational-electronic (vibronic) coupling plays a critical role in excitation energy transfer in molecular aggregates and pigment-protein complexes (PPCs). But the interplay between excitonic delocalization and vibronic interactions is complex, often leaving even qualitative questions as to what conceptual framework (e.g., Redfield versus Förster theory) should be used to interpret experimental results. To shed light on this issue, we report here on the interplay between excitonic delocalization and vibronic coupling in site-directed mutants of the water-soluble chlorophyll protein (WSCP), as reflected in 77 K fluorescence spectra. Experimentally, we find that in PPCs where excitonic delocalization is disrupted (either by mutagenesis or heterodimer formation), the relative intensity of the vibrational sideband (VSB) in fluorescence spectra is suppressed by up to 37% compared to that of the native protein. Numerical simulations reveal that this effect results from the localization of high-frequency vibrations in the coupled system; while excitonic delocalization suppresses the purely electronic transition due to H-aggregate-like dipole-dipole interference, high-frequency vibrations are unaffected, leading to a relative enhancement of the VSB. By comparing VSB intensities of PPCs both in the presence and absence of excitonic delocalization, we extract a set of "local" Huang-Rhys (HR) factors for Chl a in WSCP. More generally, our results suggest a significant role for geometric effects in controlling energy-transfer rates (which depend sensitively on absorption/fluorescence line shapes) in molecular aggregates and PPCs.

Brassica napus Drought-Induced 22-kD Protein (BnD22) Acts Simultaneously as a Cysteine Protease Inhibitor and Chlorophyll-Binding Protein.

Class II water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins that bind with chlorophyll (Chl) and its derivatives. The physiological function of WSCPs is still unclear, but it is assumed to be involved in stress responses, which is likely related to their Chl-binding and protease inhibition (PI) activities. Yet, the dual function and simultaneous functionality of WSCPs must still be better understood. Here, the biochemical functions of Brassica napus drought-induced 22-kDa protein (BnD22), a major WSCP expressed in B. napus leaves, were investigated using recombinant hexahistidine-tagged protein. We showed that BnD22 inhibited cysteine proteases, such as papain, but not serine proteases. BnD22 was able to bind with Chla or Chlb to form tetrameric complexes. Unexpectedly, BnD22-Chl tetramer displays higher inhibition toward cysteine proteases, indicating (i) simultaneous Chl-binding and PI activities and (ii) Chl-dependent activation of PI activity of BnD22. Moreover, the photostability of BnD22-Chl tetramer was reduced upon binding with the protease. Using three-dimensional structural modeling and molecular docking, we revealed that Chl binding favors interaction between BnD22 and proteases. Despite its Chl-binding ability, the BnD22 was not detected in chloroplasts but rather in the endoplasmic reticulum and vacuole. In addition, the C-terminal extension peptide of BnD22, which cleaved off post-translationally in vivo, was not implicated in subcellular localization. Instead, it drastically promoted the expression, solubility and stability of the recombinant protein.

Controlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein

Membraneless organelles (MLOs) formed via protein phase separation have great implications for both physiological and pathological processes. However, the inability to precisely control the bioactivities of MLOs has hindered our understanding of their roles in biology, not to mention their translational applications. Here, by combining intrinsically disordered domains such as RGG and mussel-foot proteins, we create an in cellulo protein phase separation system, of which various biological activities can be introduced via metal-mediated protein immobilization and further controlled by the water-soluble chlorophyll protein (WSCP)—a remarkably stable, red-light-responsive singlet oxygen generator. The WSCP-laden protein condensates undergo a liquid-to-solid phase transition on light exposure, due to oxidative crosslinking, providing a means to control catalysis within synthetic MLOs. Moreover, these photoresponsive condensates, which retain the light-induced phase-transition behavior in living cells, exhibit marked membrane localization, reminiscent of the semi-membrane-bound compartments like postsynaptic densities in nervous systems. Together, this engineered system provides an approach toward controllable synthetic MLOs and, alongside its light-induced phase transition, may well serve to emulate and explore the aging process at the subcellular or even molecular level.

Magnetophotoselection in the Investigation of Excitonically Coupled Chromophores: The Case of the Water-Soluble Chlorophyll Protein.

A magnetophotoselection (MPS) investigation of the photoexcited triplet state of chlorophyll a both in a frozen organic solvent and in a protein environment, provided by the water-soluble chlorophyll protein (WSCP) of Lepidium virginicum, is reported. The MPS experiment combines the photoselection achieved by exciting with linearly polarized light with the magnetic selection of electron paramagnetic resonance (EPR) spectroscopy, allowing the determination of the relative orientation of the optical transition dipole moment and the zero-field splitting tensor axes in both environments. We demonstrate the robustness of the proposed methodology for a quantitative description of the excitonic interactions among pigments. The orientation of the optical transition dipole moments determined by the EPR analysis in WSCP, identified as an appropriate model system, are in excellent agreement with those calculated in the point-dipole approximation. In addition, MPS provides information on the electronic properties of the triplet state, localized on a single chlorophyll a pigment of the protein cluster, in terms of orientation of the zero-field splitting tensor axes in the molecular frame.

New insights into chlorophyll-WSCP (water-soluble chlorophyll proteins) interactions : The case study of BnD22 (Brassica napus drought-induced 22 kDa)

The water-soluble chlorophyll-proteins (WSCP) of class II from Brassicaceae are non-photosynthetic proteins that bind chlorophylls (Chls) and chlorophyll derivatives. Their physiological roles, biochemical functions and mode of action are still unclear. It is assumed that the WSCPs have a protection function against Chl photodamage during stressful conditions. WSCPs are subdivided into class IIA and class IIB according to their apparent Chla/b binding ratio. Although their Chla/Chlb binding selectivity has been partly characterized, their Chl affinities are not yet precisely defined. For instance, WSCPs IIA do not show any Chl binding preference while WSCPs IIB have greater affinity to Chlb. In this study, we present a novel method for assessment of Chl binding to WSCPs based on the differences of Chl photobleaching rates in a large range of Chl/protein ratios. The protein we have chosen to study WSCP is BnD22, a WSCP IIA induced in the leaves of Brassica napus under water deficit. BnD22 formed oligomeric complexes upon binding to Chla and/or Chlb allowing a protective effect against photodamage. The binding constants indicate that BnD22 binds with high affinity the Chls and with a strong selectivity to Chla. Moreover, dependending of Chl/protein ratio upon reconstitution, two distinct binding events were detected resulting from difference of Chl stoichiometry inside oligomeric complexes.

Accurate prediction of mutation-induced frequency shifts in chlorophyll proteins with a simple electrostatic model.

Photosynthetic pigment-protein complexes control local chlorophyll (Chl) transition frequencies through a variety of electrostatic and steric forces. Site-directed mutations can modify this local spectroscopic tuning, providing critical insight into native photosynthetic functions and offering the tantalizing prospect of creating rationally designed Chl proteins with customized optical properties. Unfortunately, at present, no proven methods exist for reliably predicting mutation-induced frequency shifts in advance, limiting the method's utility for quantitative applications. Here, we address this challenge by constructing a series of pointmutants in the water-soluble chlorophyll protein of Lepidium virginicum and using them to test the reliability of a simple computational protocol for mutation-induced site energy shifts. The protocol uses molecular dynamics to prepare mutant protein structures and the charge density coupling model of Adolphs et al. [Photosynth. Res. 95, 197-209 (2008)] for site energy prediction; a graphical interface that implements the protocol automatically is published online at http://nanohub.org/tools/pigmenthunter. With the exception of a single outlier (presumably due to unexpected structural changes), we find that the calculated frequency shifts match the experiment remarkably well, with an average error of 1.6nm over a 9nm spread in wavelengths. We anticipate that the accuracy of the method can be improved in the future with more advanced sampling of mutant protein structures.

Polystyrene particles combined with di-butyl phthalate cause significant decrease in photosynthesis and red lettuce quality.

Microplastics, an emerging pollutant in the environment, have attracted extensive attention in recent years for their possible negative impact on organisms. However, direct and indirect effects of polystyrene (PS) microplastics on vegetables are still not completely known. In this study, we used red lettuce (Lactuca sativa L. Red Sails) in a hydroponic system to investigate the effects of nano- and micro-sized PS and dibutyl phthalate (DBP) on the photosynthesis and red lettuce quality. The results clearly indicated that PS reduced the bioavailability of DBP while causing a decrease in the photosynthetic parameters as well as the total chorophyll content compared to DBP alone by affecting the crystalline structure of the water-soluble chlorophyll protein. Compared with DBP monotherapy, the presence of PS significantly increased hydrogen peroxide and malondialdehyde content in the lettuce treated with DBP, indicating serious oxidative damage. Furthermore, the soluble protein and sugar content in lettuce leaves decreased with higher PS concentration and smaller PS size. It may be due to PS inhibited lettuce root and ribulose-1,5-bisphosphate carboxylase/oxygenase activities. In contrast, nitrite content increased significantly with the induction of the glutathione-ascorbic acid cycle, indicating that the presence of PS reduced the quality of DBP-treated-red lettuce. Additionally, the nano-sized PS greatly inhibited lettuce growth and quality more than the micro-sized PS. This study described the interactions between microplastics and phthalates using molecular simulation and experimental validation to highlight the potential risks of microplastics on vegetable crop production.

Excitonic Energy Level of Homo- and Hetero-Dimers and Their Composition in the Native Water-Soluble Chlorophyll Protein from Lepidium Virginicum

Abstract The native water-soluble chlorophyll protein, CP663, extracted from Lepidium virginicum, consists of four subunits, each with one chlorophyll (Chl), and the four Chls form two dimers in an orthogonal arrangement. Accordingly, CP663 is considered to have three dimers, Chl a–Chl a (AA), Chl a–Chl b (AB), and Chl b–Chl b (BB), in a certain composition and to provide six excitonic transitions because each dimer gives rise to low (L)- and high (H)-energy transitions. To investigate the excitonic transitions and the dimeric composition, the absorption and circular dichroism spectra of CP663 were measured and the electronic transitions of the dimers were calculated by Zerner’s intermediate neglect of differential overlap method. On the basis of the experimental and calculated results, the Qy-absorption band was deconvoluted by the Gaussian fitting method. The resulting six Gaussian components were assigned to the individual transitions and the excitonic transition energy was found to increase in the order AAL, ABL, AAH, BBL, ABH, and BBH. From the spectral areas of the components, the composition of the dimers was calculated as AA:AB:BB = 0.52:0.34:0.14, indicating that CP663 accommodates the homodimers in preference to the heterodimer.

How water-soluble chlorophyll protein extracts chlorophyll from membranes

Water-soluble chlorophyll proteins (WSCPs) found in Brassicaceae are non-photosynthetic proteins that bind only a small number of chlorophylls. Their biological function remains unclear, but recent data indicate that WSCPs are involved in stress response and pathogen defense as producers of reactive oxygen species and/or Chl-regulated protease inhibitors. For those functions, WSCP apoprotein supposedly binds Chl to become physiologically active or inactive, respectively. Thus, Chl-binding seems to be a pivotal step for the biological function of WSCP. WSCP can extract Chl from the thylakoid membrane but little is known about the mechanism of how Chl is sequestered from the membrane into the binding sites. Here, we investigate the interaction of WSCP with the thylakoid membrane in detail. The extraction of Chl from the thylakoid by WSCP apoprotein is a slow and inefficient reaction, because WSCP presumably does not directly extract Chl from other Chl-binding proteins embedded in the membrane. WSCP apoprotein interacts with model membranes that contain the thylakoid lipids MGDG, DGDG or PG, and can extract Chl from those. Furthermore, the WSCP-Chl complex, once formed, no longer interacts with membranes. We concluded that the surroundings of the WSCP pigment-binding site are involved in the WSCP-membrane interaction and identified a ring of hydrophobic amino acids with two conserved Trp residues around the Chl-binding site. Indeed, WSCP variants, in which one of the Trp residues was exchanged for Phe, still interact with the membrane but are no longer able to extract Chl.

Electron Nuclear Double Resonance of the Chlorophyll Triplet State in the Water-Soluble Chlorophyll Protein from Brassica oleracea: Investigation of the Effect of the Binding Site on the Hyperfine Couplings

An investigation of the photoexcited triplet state of chlorophyll (Chl) a in the water-soluble chlorophyll protein (WSCP) of Brassica oleracea has been carried out by means of electron-nuclear double resonance (ENDOR), achieving a complete assignment of the observed hyperfine couplings corresponding to methine protons and methyl groups of Chl a triplet state. The triplet-state properties, and in particular the hyperfine couplings, were found to be similar to those previously reported for Chl a in the WSCP of Lepidium virginicum. Therefore, the porphyrin ring deformation observed in Brassica oleracea WSCP seems to only slightly affect the spin density of 3Chl a. This may be relevant when considering the robustness of triplet–triplet energy transfer mechanisms, relying on wavefunction overlap, in systems, such as the photosynthetic light-harvesting complexes, in which Chl triplet states with distorted geometries are involved.

The pigment binding behaviour of water-soluble chlorophyll protein (WSCP).

Water-soluble chlorophyll proteins (WSCPs) are homotetrameric proteins that bind four chlorophyll (Chl) molecules in identical binding sites, which makes WSCPs a good model to study protein-pigment interactions. In a previous study, we described preferential binding of Chl a or Chl b in various WSCP versions. Chl b binding is preferred when a hydrogen bond can be formed between the C7 formyl of the chlorin macrocycle and the protein, whereas Chl a is preferred when Chl b binding is sterically unfavorable. Here, we determined the binding affinities and kinetics of various WSCP versions not only for Chl a/b, but also for chlorophyllide (Chlide) a/b and pheophytin (Pheo) a/b. Altered KD values are responsible for the Chl a/b selectivity in WSCP whereas differences in the reaction kinetics are neglectable in explaining different Chl a/b preferences. WSCP binds both Chlide and Pheo with a lower affinity than Chl, which indicates the importance of the phytol chain and the central Mg2+ ion as interaction sites between WSCP and pigment. Pheophorbide (Pheoide), lacking both the phytol chain and the central Mg2+ ion, can only be bound as Pheoide b to a WSCP that has a higher affinity for Chl b than Chl a, which underlines the impact of the C7 formyl-protein interaction. Moreover, WSCP was able to bind protochlorophyllide and Mg-protoporphyrin IX, which suggests that neither the size of the π electron system of the macrocycle nor the presence of a fifth ring at the macrocycle notably affect the binding to WSCP. WSCP also binds heme to form a tetrameric complex, suggesting that heme is bound in the Chl-binding site.

How the Protein Environment Can Tune the Energy, the Coupling, and the Ultrafast Dynamics of Interacting Chlorophylls: The Example of the Water-Soluble Chlorophyll Protein.

The interplay between active molecules and the protein environment in light-harvesting complexes tunes the photophysics and the dynamical properties of pigment–protein complexes in a subtle way, which is not fully understood. Here we characterized the photophysics and the ultrafast dynamics of four variants of the water-soluble chlorophyll protein (WSCP) as an ideal model system to study the behavior of strongly interacting chlorophylls. We found that when coordinated by the WSCP protein, the presence of the formyl group in chlorophyll b replacing the methyl group in chlorophyll a strongly affects the exciton energy and the dynamics of the system, opening up the possibility of tuning the photophysics and the transport properties of multichromophores by engineering specific interactions with the surroundings.

How water-mediated hydrogen bonds affect chlorophyll a/b selectivity in Water-Soluble Chlorophyll Protein

The Water-Soluble Chlorophyll Protein (WSCP) of Brassicaceae is a remarkably stable tetrapyrrole-binding protein that, by virtue of its simple design, is an exceptional model to investigate the interactions taking place between pigments and their protein scaffold and how they affect the photophysical properties and the functionality of the complexes. We investigated variants of WSCP from Lepidium virginicum (Lv) and Brassica oleracea (Bo), reconstituted with Chlorophyll (Chl) b, to determine the mechanisms by which the different Chl binding sites control their Chl a/b specificities. A combined Raman and crystallographic investigation has been employed, aimed to characterize in detail the hydrogen-bond network involving the formyl group of Chl b. The study revealed a variable degree of conformational freedom of the hydrogen bond networks among the WSCP variants, and an unexpected mixed presence of hydrogen-bonded and not hydrogen-bonded Chls b in the case of the L91P mutant of Lv WSCP. These findings helped to refine the description of the mechanisms underlying the different Chl a/b specificities of WSCP versions, highlighting the importance of the structural rigidity of the Chl binding site in the vicinity of the Chl b formyl group in granting a strong selectivity to binding sites.

Stability of Water-Soluble Chlorophyll Protein (WSCP) Depends on Phytyl Conformation.

Water-soluble chlorophyll proteins (WSCP) from Brassicaceae form homotetrameric chlorophyll (Chl)–protein complexes binding one Chl per apoprotein and no carotenoids. Despite the lack of photoprotecting pigments, the complex-bound Chls displays a remarkable stability toward photodynamic damage. On the basis of a mutational study, we show that not only the presence of the phytyls is necessary for photoprotection in WSCPs, as we previously demonstrated, but also is their correct conformation and localization. The extreme heat stability of WSCP also depends on the presence of the phytyl chains, confirming their relevance for the unusual stability of WSCP.

Chlorophyll a/b binding-specificity in water-soluble chlorophyll protein.

We altered the chlorophyll (Chl) binding sites in various versions of water-soluble chlorophyll protein (WSCP) by amino acid exchanges to alter their preferences for either Chl a or Chl b. WSCP is ideally suited for this mutational analysis since it forms a tetrameric complex with only four identical Chl binding sites. A loop of 4-6 amino acids is responsible for Chl a versus Chl b selectivity. We show that a single amino acid exchange within this loop changes the relative Chl a/b affinities by a factor of 40. We obtained crystal structures of this WSCP variant binding either Chl a or Chl b. The Chl binding sites in these structures were compared with those in the major light-harvesting complex (LHCII) of the photosynthetic apparatus in plants to search for similar structural features involved in Chl a/b binding specificity.

Optically Detected Magnetic Resonance of Chlorophyll Triplet States in Water-Soluble Chlorophyll Proteins from Lepidium virginicum: Evidence for Excitonic Interaction among the Four Pigments.

Optically detected magnetic resonance of triplet states populated by photoexcitation in water-soluble chlorophyll proteins (WSCPs) from Lepidium virginicum has been performed using both absorption and fluorescence detection. Well resolved triplet-singlet (T-S) spectra have been obtained and interpreted in terms of electronic interactions among the four chlorophylls (Chls), forming two dimers in the WSCP tetramer. Localization of the triplet state on a single Chl leads to a redistribution of the oscillator strength in the remaining three Chls of the complex. By comparing the spectra with those obtained on a substoichiometric WSCP complex containing only 2 Chls per protein tetramer, we proved that, to interpret the optical spectra of the WSCP fully loaded with 4 Chls, the interactions between the two dimers have to be taken into account and cannot be considered negligible. The results show that the WSCP may well be considered as an ideal model system to study Chl-Chl interactions, also in view of the possibility to modify the number and molecular structure of the bound porphyrin chromophores.

Accessibility of Protein-Bound Chlorophylls Probed by Dynamic Electron Polarization.

The possibility to probe the accessibility of sites of proteins represents an important point to explore their interactions with specific substrates in solution. The dynamic electron polarization of nitroxide radicals induced by excited triplet states of organic molecules is a phenomenon that is known to occur in aqueous solutions. The interaction within the radical-triplet pair causes a net emissive dynamic electron polarization of the nitroxide radical, that can be detected by means of time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy. We have exploited this effect to prove the accessibility of chlorophylls bound to a protein, namely, the water-soluble chlorophyll protein WSCP. The results have important implications for topological studies in macromolecules.

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.

Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls.

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

Biosynthesis of Chlorophyll a in a Purple Bacterial Phototroph and Assembly into a Plant Chlorophyll-Protein Complex.

Improvements to photosynthetic efficiency could be achieved by manipulating pigment biosynthetic pathways of photosynthetic organisms in order to increase the spectral coverage for light absorption. The development of organisms that can produce both bacteriochlorophylls and chlorophylls is one way to achieve this aim, and accordingly we have engineered the bacteriochlorophyll-utilizing anoxygenic phototroph Rhodobacter sphaeroides to make chlorophyll a. Bacteriochlorophyll and chlorophyll share a common biosynthetic pathway up to the precursor chlorophyllide. Deletion of genes responsible for the bacteriochlorophyll-specific modifications of chlorophyllide and replacement of the native bacteriochlorophyll synthase with a cyanobacterial chlorophyll synthase resulted in the production of chlorophyll a. This pigment could be assembled in vivo into the plant water-soluble chlorophyll protein, heterologously produced in Rhodobacter sphaeroides, which represents a proof-of-principle for the engineering of novel antenna complexes that enhance the spectral range of photosynthesis.