From isolated light-harvesting complexes to the thylakoid membrane: a single-molecule perspective

AbstractThe conversion of solar radiation to chemical energy in plants and green algae takes place in the thylakoid membrane. This amphiphilic environment hosts a complex arrangement of light-harvesting pigment-protein complexes that absorb light and transfer the excitation energy to photochemically active reaction centers. This efficient light-harvesting capacity is moreover tightly regulated by a photoprotective mechanism called non-photochemical quenching to avoid the stress-induced destruction of the catalytic reaction center. In this review we provide an overview of single-molecule fluorescence measurements on plant light-harvesting complexes (LHCs) of varying sizes with the aim of bridging the gap between the smallest isolated complexes, which have been well-characterized, and the native photosystem. The smallest complexes contain only a small number (10–20) of interacting chlorophylls, while the native photosystem contains dozens of protein subunits and many hundreds of connected pigments. We discuss the functional significance of conformational dynamics, the lipid environment, and the structural arrangement of this fascinating nano-machinery. The described experimental results can be utilized to build mathematical-physical models in a bottom-up approach, which can then be tested on larger in vivo systems. The results also clearly showcase the general property of biological systems to utilize the same system properties for different purposes. In this case it is the regulated conformational flexibility that allows LHCs to switch between efficient light-harvesting and a photoprotective function.

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Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane

Life ◽

10.3390/life11010015 ◽

2020 ◽

Vol 11 (1) ◽

pp. 15

Author(s):

Radek Kaňa ◽

Gábor Steinbach ◽

Roman Sobotka ◽

György Vámosi ◽

Josef Komenda

Keyword(s):

Membrane Proteins ◽

Single Molecule ◽

Protein Interactions ◽

Thylakoid Membrane ◽

Protein Complexes ◽

Correlation Spectroscopy ◽

Membrane Microdomains ◽

Fast Diffusion ◽

Light Harvesting Complexes ◽

Term Survival

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

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Identification of parallel pH- and zeaxanthin-dependent quenching of excess energy in LHCSR3 in Chlamydomonas reinhardtii

10.1101/2020.07.10.197483 ◽

2020 ◽

Author(s):

Julianne M. Troiano ◽

Federico Perozeni ◽

Raymundo Moya ◽

Luca Zuliani ◽

Kwangryul Baek ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Light Harvesting ◽

Complex Stress ◽

Excess Energy ◽

Photochemical Quenching ◽

Light Harvesting Complexes ◽

Light Harvesting Complex ◽

Non Photochemical Quenching

AbstractUnder high light conditions, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating excess absorbed energy, which is called non-photochemical quenching (NPQ). In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via pH and serves as a quenching site. However, the mechanisms by which LHCSR3 functions have not been determined. Using a combined in vivo and in vitro approach, we identify two parallel yet distinct quenching processes, individually controlled by pH and carotenoid composition, and their likely molecular origin within LHCSR3 from Chlamydomonas reinhardtii. The pH-controlled quenching is removed within a mutant LHCSR3 that lacks the protonable residues responsible for sensing pH. Constitutive quenching in zeaxanthin-enriched systems demonstrates zeaxanthin-controlled quenching, which may be shared with other light-harvesting complexes. We show that both quenching processes prevent the formation of damaging reactive oxygen species, and thus provide distinct timescales and mechanisms of protection in a changing environment.

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Optimization of light harvesting and photoprotection: molecular mechanisms and physiological consequences

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2012.0069 ◽

2012 ◽

Vol 367 (1608) ◽

pp. 3455-3465 ◽

Cited By ~ 76

Author(s):

Peter Horton

Keyword(s):

Protein Interactions ◽

Thylakoid Membrane ◽

Molecular Mechanisms ◽

Light Harvesting ◽

Protein Complexes ◽

Efficient Energy Transfer ◽

Light Harvesting Complexes ◽

Functional Flexibility ◽

Jan Anderson ◽

Repulsive Forces

The distinctive lateral organization of the protein complexes in the thylakoid membrane investigated by Jan Anderson and co-workers is dependent on the balance of various attractive and repulsive forces. Modulation of these forces allows critical physiological regulation of photosynthesis that provides efficient light-harvesting in limiting light but dissipation of excess potentially damaging radiation in saturating light. The light-harvesting complexes (LHCII) are central to this regulation, which is achieved by phosphorylation of stromal residues, protonation on the lumen surface and de-epoxidation of bound violaxanthin. The functional flexibility of LHCII derives from a remarkable pigment composition and configuration that not only allow efficient absorption of light and efficient energy transfer either to photosystem II or photosystem I core complexes, but through subtle configurational changes can also exhibit highly efficient dissipative reactions involving chlorophyll–xanthophyll and/or chlorophyll–chlorophyll interactions. These changes in function are determined at a macroscopic level by alterations in protein–protein interactions in the thylakoid membrane. The capacity and dynamics of this regulation are tuned to different physiological scenarios by the exact protein and pigment content of the light-harvesting system. Here, the molecular mechanisms involved will be reviewed, and the optimization of the light-harvesting system in different environmental conditions described.

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The lutein epoxide cycle in higher plants: its relationships to other xanthophyll cycles and possible functions

Functional Plant Biology ◽

10.1071/fp07095 ◽

2007 ◽

Vol 34 (9) ◽

pp. 759 ◽

Cited By ~ 88

Author(s):

Jose I. García-Plazaola ◽

Shizue Matsubara ◽

C. Barry Osmond

Keyword(s):

Light Harvesting ◽

Higher Plants ◽

Protein Complexes ◽

Light Exposure ◽

Photosynthetic Pigment ◽

In Vivo Studies ◽

Photochemical Quenching ◽

Strong Light

Several xanthophyll cycles have been described in photosynthetic organisms. Among them, only two are present in higher plants: the ubiquitous violaxanthin (V) cycle, and the taxonomically restricted lutein epoxide (Lx) cycle, whereas four cycles seem to occur in algae. Although V is synthesised through the β-branch of the carotenoid biosynthetic pathway and Lx is the product of the α-branch; both are co-located in the same sites of the photosynthetic pigment-protein complexes isolated from thylakoids. Both xanthophylls are also de-epoxidised upon light exposure by the same enzyme, violaxanthin de-epoxidase (VDE) leading to the formation of zeaxanthin (Z) and lutein (L) at comparable rates. In contrast with VDE, the reverse reaction presumably catalysed by zeaxanthin epoxidase (ZE), is much slower (or even inactive) with L than with antheraxanthin (A) or Z. Consequently many species lack Lx altogether, and although the presence of Lx shows an irregular taxonomical distribution in unrelated taxa, it has a high fidelity at family level. In those plants which accumulate Lx, variations in ZE activity in vivo mean that a complete Lx-cycle occurs in some (with Lx pools being restored overnight), whereas in others a truncated cycle is observed in which VDE converts Lx into L, but regeneration of Lx by ZE is extremely slow. Accumulation of Lx to high concentrations is found most commonly in old leaves in deeply shaded canopies, and the Lx cycle in these leaves is usually truncated. This seemingly anomalous situation presumably arises because ZE has a low but finite affinity for L, and because deeply shaded leaves are not often exposed to light intensities strong enough to activate VDE. Notably, both in vitro and in vivo studies have recently shown that accumulation of Lx can increase the light harvesting efficiency in the antennae of PSII. We propose a model for the truncated Lx cycle in strong light in which VDE converts Lx to L which then occupies sites L2 and V1 in the light-harvesting antenna complex of PSII (Lhcb), displacing V and Z. There is correlative evidence that this photoconverted L facilitates energy dissipation via non-photochemical quenching and thereby converts a highly efficient light harvesting system to an energy dissipating system with improved capacity to engage photoprotection. Operation of the α- and β-xanthophyll cycles with different L and Z epoxidation kinetics thus allows a combination of rapidly and slowly reversible modulation of light harvesting and photoprotection, with each cycle having distinct effects. Based on the patchy taxonomical distribution of Lx, we propose that the presence of Lx (and the Lx cycle) could be the result of a recurrent mutation in the epoxidase gene that increases its affinity for L, which is conserved whenever it confers an evolutionary advantage.

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Metal-Enhanced Fluorescence of Chlorophylls in Single Light-Harvesting Complexes

MRS Proceedings ◽

10.1557/proc-1208-o14-01 ◽

2009 ◽

Vol 1208 ◽

Author(s):

Sebastian Mackowski ◽

Dawid Piatkowski ◽

Stephan Wörmke ◽

Achim Hartschuh ◽

Christoph Bräeuchle ◽

...

Keyword(s):

Single Molecule ◽

Light Harvesting ◽

Protein Complexes ◽

Fluorescence Emission ◽

Fold Increase ◽

Metal Nanoparticle ◽

Metal Nanostructures ◽

Enhanced Fluorescence ◽

Light Harvesting Complexes ◽

Light Harvesting Complex

AbstractWe show that the fluorescence of peridinin-chlorophyll a-protein complexes can be strongly enhanced via coupling with plasmon excitations localized in metal nanostructures. The results of ensemble and single-molecule spectroscopy experiments at room temperature demonstrate six-fold increase of the emission intensity of the light-harvesting complex when it is placed in the vicinity of chemically prepared silver islands. Irrespective of the enhancement, we observe no effect of the metal nanoparticle on the fluorescence emission energy of the complex. This observation implies that plasmon excitations may be applied for controlling the optical properties of complex biomolecules.

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Conformational entropy of a single peptide controlled under force governs protease recognition and catalysis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803872115 ◽

2018 ◽

Vol 115 (45) ◽

pp. 11525-11530 ◽

Cited By ~ 4

Author(s):

Marcelo E. Guerin ◽

Guillaume Stirnemann ◽

David Giganti

Keyword(s):

Single Molecule ◽

Conformational Dynamics ◽

Conformational Sampling ◽

Enzymatic Reactions ◽

Sequence Specificity ◽

Proteomics Data ◽

Conformational Entropy ◽

Substrate Peptide ◽

The Impact

An immense repertoire of protein chemical modifications catalyzed by enzymes is available as proteomics data. Quantifying the impact of the conformational dynamics of the modified peptide remains challenging to understand the decisive kinetics and amino acid sequence specificity of these enzymatic reactions in vivo, because the target peptide must be disordered to accommodate the specific enzyme-binding site. Here, we were able to control the conformation of a single-molecule peptide chain by applying mechanical force to activate and monitor its specific cleavage by a model protease. We found that the conformational entropy impacts the reaction in two distinct ways. First, the flexibility and accessibility of the substrate peptide greatly increase upon mechanical unfolding. Second, the conformational sampling of the disordered peptide drives the specific recognition, revealing force-dependent reaction kinetics. These results support a mechanism of peptide recognition based on conformational selection from an ensemble that we were able to quantify with a torsional free-energy model. Our approach can be used to predict how entropy affects site-specific modifications of proteins and prompts conformational and mechanical selectivity.

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Microscopic quantum coherence in a photosynthetic-light-harvesting antenna

Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences ◽

10.1098/rsta.2011.0207 ◽

2012 ◽

Vol 370 (1972) ◽

pp. 3672-3691 ◽

Cited By ~ 28

Author(s):

Jahan M. Dawlaty ◽

Akihito Ishizaki ◽

Arijit K. De ◽

Graham R. Fleming

Keyword(s):

Single Molecule ◽

Quantum Coherence ◽

Energy Transport ◽

Light Harvesting ◽

Electronic Spectroscopy ◽

Coherent Motion ◽

Quantum Beats ◽

Ensemble Averaging ◽

Light Harvesting Antenna

We briefly review the coherent quantum beats observed in recent two-dimensional electronic spectroscopy experiments in a photosynthetic-light-harvesting antenna. We emphasize that the decay of the quantum beats in these experiments is limited by ensemble averaging. The in vivo dynamics of energy transport depends upon the local fluctuations of a single photosynthetic complex during the energy transfer time (a few picoseconds). Recent analyses suggest that it remains possible that the quantum-coherent motion may be robust under individual realizations of the environment-induced fluctuations contrary to intuition obtained from condensed phase spectroscopic measurements and reduced density matrices. This result indicates that the decay of the observed quantum coherence can be understood as ensemble dephasing. We propose a fluorescence-detected single-molecule experiment with phase-locked excitation pulses to investigate the coherent dynamics at the level of a single molecule without hindrance by ensemble averaging. We discuss the advantages and limitations of this method. We report our initial results on bulk fluorescence-detected coherent spectroscopy of the Fenna–Mathews–Olson complex.

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Structure of the stress-related LHCSR1 complex determined by an integrated computational strategy

10.1101/2021.10.06.463383 ◽

2021 ◽

Author(s):

Ingrid Guarnetti Prandi ◽

Vladislav Sláma ◽

Cristina Pecorilla ◽

Lorenzo Cupellini ◽

Benedetta Mennucci

Keyword(s):

Structural Model ◽

Light Harvesting ◽

Structural Information ◽

Protein Complexes ◽

Complex Stress ◽

Main Function ◽

Light Harvesting Complexes ◽

Light Harvesting Complex ◽

Pigment Protein Complexes ◽

Computational Strategy

Light-harvesting complexes (LHCs) are pigment-protein complexes whose main function is to capture sunlight and transfer the energy to reaction centers of photosystems. In response to varying light conditions, LH complexes also play photoregulation and photoprotection roles. In algae and mosses, a sub-family of LHCs, Light-Harvesting complex stress related (LHCSR), is responsible for photoprotective quenching. Despite their functional and evolutionary importance, no direct structural information on LHCSRs is available that can explain their unique properties. In this work we propose a structural model of LHCSR1 from the moss P. Patens, obtained through an integrated computational strategy that combines homology modeling, molecular dynamics, and multiscale quantum chemical calculations. The model is validated by reproducing the spectral properties of LHCSR1. Our model reveals the structural specificity of LHCSR1, as compared with the CP29 LH complex, and poses the basis for understanding photoprotective quenching in mosses.

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Contribution of low-temperature single-molecule techniques to structural issues of pigment–protein complexes from photosynthetic purple bacteria

Journal of The Royal Society Interface ◽

10.1098/rsif.2017.0680 ◽

2018 ◽

Vol 15 (138) ◽

pp. 20170680 ◽

Cited By ~ 3

Author(s):

Alexander Löhner ◽

Richard Cogdell ◽

Jürgen Köhler

Keyword(s):

Low Temperature ◽

Single Molecule ◽

Degrees Of Freedom ◽

Protein Complexes ◽

Purple Bacteria ◽

Structural Models ◽

Spectral Features ◽

Light Harvesting Complexes ◽

Pigment Protein Complexes ◽

Photosynthetic Purple Bacteria

As the electronic energies of the chromophores in a pigment–protein complex are imposed by the geometrical structure of the protein, this allows the spectral information obtained to be compared with predictions derived from structural models. Thereby, the single-molecule approach is particularly suited for the elucidation of specific, distinctive spectral features that are key for a particular model structure, and that would not be observable in ensemble-averaged spectra due to the heterogeneity of the biological objects. In this concise review, we illustrate with the example of the light-harvesting complexes from photosynthetic purple bacteria how results from low-temperature single-molecule spectroscopy can be used to discriminate between different structural models. Thereby the low-temperature approach provides two advantages: (i) owing to the negligible photobleaching, very long observation times become possible, and more importantly, (ii) at cryogenic temperatures, vibrational degrees of freedom are frozen out, leading to sharper spectral features and in turn to better resolved spectra.

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