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1.
Lab Chip ; 20(14): 2529-2538, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32662473

RESUMO

Light-harvesting 2 (LH2) and light-harvesting 1 - reaction centre (RCLH1) complexes purified from the photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were cross-patterned on glass surfaces for energy transfer studies. Atomic force microscopy (AFM) images of the RCLH1 and LH2 patterns show the deposition of monomolecular layers of complexes on the glass substrate. Spectral imaging and fluorescence life-time imaging microscopy (FLIM) revealed that RCLH1 and LH2 complexes, sealed under physiological conditions, retained their native light-harvesting and energy transfer functions. Measurements of the amplitude and lifetime decay of fluorescence emission from LH2 complexes, the energy transfer donors, and gain of fluorescence emission from acceptor RCLH1 complexes, provide evidence for excitation energy transfer from LH2 to RCLH1. Directional energy transfer on the glass substrate was unequivocally established by using LH2-carotenoid complexes and RCLH1 complexes with genetically removed carotenoids. Specific excitation of carotenoids in donor LH2 complexes elicited fluorescence emission from RCLH1 acceptors. To explore the longevity of this novel nanoprinted photosynthetic unit, RCLH1 and LH2 complexes were cross-patterned on a glass surface and sealed under a protective argon atmosphere. The results show that both complexes retained their individual and collective functions and are capable of directional excitation energy transfer for at least 60 days.

2.
J Am Chem Soc ; 142(32): 13898-13907, 2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32672948

RESUMO

Advances in protein design and engineering have yielded peptide assemblies with enhanced and non-native functionalities. Here, various molecular organic semiconductors (OSCs), with known excitonic up- and down-conversion properties, are attached to a de novo-designed protein, conferring entirely novel functions on the peptide scaffolds. The protein-OSC complexes form similarly sized, stable, water-soluble nanoparticles that are robust to cryogenic freezing and processing into the solid-state. The peptide matrix enables the formation of protein-OSC-trehalose glasses that fix the proteins in their folded states under oxygen-limited conditions. The encapsulation dramatically enhances the stability of protein-OSC complexes to photodamage, increasing the lifetime of the chromophores from several hours to more than 10 weeks under constant illumination. Comparison of the photophysical properties of astaxanthin aggregates in mixed-solvent systems and proteins shows that the peptide environment does not alter the underlying electronic processes of the incorporated materials, exemplified here by singlet exciton fission followed by separation into weakly bound, localized triplets. This adaptable protein-based approach lays the foundation for spectroscopic assessment of a broad range of molecular OSCs in aqueous solutions and the solid-state, circumventing the laborious procedure of identifying the experimental conditions necessary for aggregate generation or film formation. The non-native protein functions also raise the prospect of future biocompatible devices where peptide assemblies could complex with native and non-native systems to generate novel functional materials.

3.
Biochem J ; 477(12): 2313-2325, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32469391

RESUMO

The unique isocyclic E ring of chlorophylls contributes to their role as light-absorbing pigments in photosynthesis. The formation of the E ring is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase, and the O2-dependent cyclase in prokaryotes consists of a diiron protein AcsF, augmented in cyanobacteria by an auxiliary subunit Ycf54. Here, we establish the composition of plant and algal cyclases, by demonstrating the in vivo heterologous activity of O2-dependent cyclases from the green alga Chlamydomonas reinhardtii and the model plant Arabidopsis thaliana in the anoxygenic photosynthetic bacterium Rubrivivax gelatinosus and in the non-photosynthetic bacterium Escherichia coli. In each case, an AcsF homolog is the core catalytic subunit, but there is an absolute requirement for an algal/plant counterpart of Ycf54, so the necessity for an auxiliary subunit is ubiquitous among oxygenic phototrophs. A C-terminal ∼40 aa extension, which is present specifically in green algal and plant Ycf54 proteins, may play an important role in the normal function of the protein as a cyclase subunit.

4.
J Biol Chem ; 295(20): 6888-6925, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32241908

RESUMO

Modified tetrapyrroles are large macrocyclic compounds, consisting of diverse conjugation and metal chelation systems and imparting an array of colors to the biological structures that contain them. Tetrapyrroles represent some of the most complex small molecules synthesized by cells and are involved in many essential processes that are fundamental to life on Earth, including photosynthesis, respiration, and catalysis. These molecules are all derived from a common template through a series of enzyme-mediated transformations that alter the oxidation state of the macrocycle and also modify its size, its side-chain composition, and the nature of the centrally chelated metal ion. The different modified tetrapyrroles include chlorophylls, hemes, siroheme, corrins (including vitamin B12), coenzyme F430, heme d 1, and bilins. After nearly a century of study, almost all of the more than 90 different enzymes that synthesize this family of compounds are now known, and expression of reconstructed operons in heterologous hosts has confirmed that most pathways are complete. Aside from the highly diverse nature of the chemical reactions catalyzed, an interesting aspect of comparative biochemistry is to see how different enzymes and even entire pathways have evolved to perform alternative chemical reactions to produce the same end products in the presence and absence of oxygen. Although there is still much to learn, our current understanding of tetrapyrrole biogenesis represents a remarkable biochemical milestone that is summarized in this review.

5.
Proc Natl Acad Sci U S A ; 117(12): 6752-6761, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32144140

RESUMO

A type of chromosome-free cell called SimCells (simple cells) has been generated from Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome.


Assuntos
Antineoplásicos/farmacologia , Catecóis/farmacologia , Reprogramação Celular , Cupriavidus necator/genética , Escherichia coli/genética , Pseudomonas putida/genética , Biologia Sintética/métodos , Proliferação de Células , Cromossomos Bacterianos , Cupriavidus necator/metabolismo , Sistemas de Liberação de Medicamentos , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Engenharia Genética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Pseudomonas putida/metabolismo , Células Tumorais Cultivadas
6.
Proc Natl Acad Sci U S A ; 117(12): 6502-6508, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32139606

RESUMO

Carotenoids play a number of important roles in photosynthesis, primarily providing light-harvesting and photoprotective energy dissipation functions within pigment-protein complexes. The carbon-carbon double bond (C=C) conjugation length of carotenoids (N), generally between 9 and 15, determines the carotenoid-to-(bacterio)chlorophyll [(B)Chl] energy transfer efficiency. Here we purified and spectroscopically characterized light-harvesting complex 2 (LH2) from Rhodobacter sphaeroides containing the N = 7 carotenoid zeta (ζ)-carotene, not previously incorporated within a natural antenna complex. Transient absorption and time-resolved fluorescence show that, relative to the lifetime of the S1 state of ζ-carotene in solvent, the lifetime decreases ∼250-fold when ζ-carotene is incorporated within LH2, due to transfer of excitation energy to the B800 and B850 BChls a These measurements show that energy transfer proceeds with an efficiency of ∼100%, primarily via the S1 → Qx route because the S1 → S0 fluorescence emission of ζ-carotene overlaps almost perfectly with the Qx absorption band of the BChls. However, transient absorption measurements performed on microsecond timescales reveal that, unlike the native N ≥ 9 carotenoids normally utilized in light-harvesting complexes, ζ-carotene does not quench excited triplet states of BChl a, likely due to elevation of the ζ-carotene triplet energy state above that of BChl a These findings provide insights into the coevolution of photosynthetic pigments and pigment-protein complexes. We propose that the N ≥ 9 carotenoids found in light-harvesting antenna complexes represent a vital compromise that retains an acceptable level of energy transfer from carotenoids to (B)Chls while allowing acquisition of a new, essential function, namely, photoprotective quenching of harmful (B)Chl triplets.


Assuntos
Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Carotenoides/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Proteínas de Bactérias/química , Carotenoides/química , Transferência de Energia , Cinética , Complexos de Proteínas Captadores de Luz/química , Fotossíntese , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/metabolismo
7.
Photosynth Res ; 144(2): 155-169, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31350671

RESUMO

Six variants of the LH2 antenna complex from Rba. sphaeroides, comprising the native B800-B850, B800-free LH2 (B850) and four LH2s with various (bacterio)chlorophylls reconstituted into the B800 site, have been investigated with static and time-resolved optical spectroscopies at room temperature and at 77 K. The study particularly focused on how reconstitution of a non-native (bacterio)chlorophylls affects excitation energy transfer between the naturally bound carotenoid spheroidene and artificially substituted pigments in the B800 site. Results demonstrate there is no apparent trend in the overall energy transfer rate from spheroidene to B850 bacteriochlorophyll a; however, a trend in energy transfer rate from the spheroidene S1 state to Qy of the B800 (bacterio)chlorophylls is noticeable. These outcomes were applied to test the validity of previously proposed energy values of the spheroidene S1 state, supporting a value in the vicinity of 13,400 cm-1 (746 nm).

8.
Biochim Biophys Acta Bioenerg ; 1861(4): 148064, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421078

RESUMO

Some cyanobacteria remodel their photosynthetic apparatus by a process known as Far-Red Light Photoacclimation (FaRLiP). Specific subunits of the phycobilisome (PBS), photosystem I (PSI), and photosystem II (PSII) complexes produced in visible light are replaced by paralogous subunits encoded within a conserved FaRLiP gene cluster when cells are grown in far-red light (FRL; λ = 700-800 nm). FRL-PSII complexes from the FaRLiP cyanobacterium, Synechococcus sp. PCC 7335, were purified and shown to contain Chl a, Chl d, Chl f, and pheophytin a, while FRL-PSI complexes contained only Chl a and Chl f. The spectroscopic properties of purified photosynthetic complexes from Synechococcus sp. PCC 7335 were determined individually, and energy transfer kinetics among PBS, PSII, and PSI were analyzed by time-resolved fluorescence (TRF) spectroscopy. Direct energy transfer from PSII to PSI was observed in cells (and thylakoids) grown in red light (RL), and possible routes of energy transfer in both RL- and FRL-grown cells were inferred. Three structural arrangements for RL-PSI were observed by atomic force microscopy of thylakoid membranes, but only arrays of trimeric FRL-PSI were observed in thylakoids from FRL-grown cells. Cells grown in FRL synthesized the FRL-specific complexes but also continued to synthesize some PBS and PSII complexes identical to those produced in RL. Although the light-harvesting efficiency of photosynthetic complexes produced in FRL might be lower in white light than the complexes produced in cells acclimated to white light, the FRL-complexes provide cells with the flexibility to utilize both visible and FRL to support oxygenic photosynthesis. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.


Assuntos
Aclimatação/efeitos da radiação , Transferência de Energia/efeitos da radiação , Luz , Fotossíntese/efeitos da radiação , Synechococcus/fisiologia , Clorofila/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Espectrometria de Fluorescência , Synechococcus/efeitos da radiação
9.
Microb Biotechnol ; 13(2): 363-367, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31880868

RESUMO

Cyanobacteria are prokaryotic phototrophs that, in addition to being excellent model organisms for studying photosynthesis, have tremendous potential for light-driven synthetic biology and biotechnology. These versatile and resilient microorganisms harness the energy of sunlight to oxidise water, generating chemical energy (ATP) and reductant (NADPH) that can be used to drive sustainable synthesis of high-value natural products in genetically modified strains. In this commentary article for the Synthetic Microbiology Caucus we discuss the great progress that has been made in engineering cyanobacterial hosts as microbial cell factories for solar-powered biosynthesis. We focus on some of the main areas where the synthetic biology and metabolic engineering tools in cyanobacteria are not as advanced as those in more widely used heterotrophic chassis, and go on to highlight key improvements that we feel are required to unlock the full power of cyanobacteria for future green biotechnology.

10.
Nature ; 575(7783): 535-539, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723268

RESUMO

The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.


Assuntos
Microscopia Crioeletrônica , Complexo Citocromos b6f/química , Complexo Citocromos b6f/ultraestrutura , Spinacia oleracea/química , Spinacia oleracea/ultraestrutura , Sítios de Ligação , Clorofila/química , Heme/química , Lipídeos/química , Modelos Moleculares , Oxirredução , Fotossíntese , Plastoquinona/química , Relação Estrutura-Atividade
11.
Cell ; 179(5): 1098-1111.e23, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31730852

RESUMO

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

12.
Appl Environ Microbiol ; 86(1)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31653788

RESUMO

Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans.IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.

13.
Plant Cell ; 31(12): 2912-2928, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31615847

RESUMO

The membrane-embedded FtsH proteases found in bacteria, chloroplasts, and mitochondria are involved in diverse cellular processes including protein quality control and regulation. The genome of the model cyanobacterium Synechocystis sp PCC 6803 encodes four FtsH homologs designated FtsH1 to FtsH4. The FtsH3 homolog is present in two hetero-oligomeric complexes: FtsH2/3, which is responsible for photosystem II quality control, and the essential FtsH1/3 complex, which helps maintain Fe homeostasis by regulating the level of the transcription factor Fur. To gain a more comprehensive insight into the physiological roles of FtsH hetero-complexes, we performed genome-wide expression profiling and global proteomic analyses of Synechocystis mutants conditionally depleted of FtsH3 or FtsH1 grown under various nutrient conditions. We show that the lack of FtsH1/3 leads to a drastic reduction in the transcriptional response to nutrient stress of not only Fur but also the Pho, NdhR, and NtcA regulons. In addition, this effect is accompanied by the accumulation of the respective transcription factors. Thus, the FtsH1/3 complex is of critical importance for acclimation to iron, phosphate, carbon, and nitrogen starvation in Synechocystis.plantcell;31/12/2912/FX1F1fx1.

14.
Biochem J ; 476(15): 2173-2190, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31320503

RESUMO

The reversible docking of small, diffusible redox proteins onto a membrane protein complex is a common feature of bacterial, mitochondrial and photosynthetic electron transfer (ET) chains. Spectroscopic studies of ensembles of such redox partners have been used to determine ET rates and dissociation constants. Here, we report a single-molecule analysis of the forces that stabilise transient ET complexes. We examined the interaction of two components of bacterial photosynthesis, cytochrome c 2 and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC-LH1-PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome c 2 molecules. Microscale patterns of cytochrome c 2 and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome c 2 Following the transient association of photo-oxidised RC and reduced cytochrome c 2 molecules, retraction of the RC-functionalised probe met with resistance, and forces between 112 and 887 pN were required to disrupt the post-ET RC-c 2 complex, depending on the retraction velocities used. If tip-attached RCs were reduced instead, the probability of interaction with reduced cytochrome c 2 molecules decreased 5-fold. Thus, the redox states of the cytochrome c 2 haem cofactor and RC 'special pair' bacteriochlorophyll dimer are important for establishing a productive ET complex. The millisecond persistence of the post-ET cytochrome c 2[oxidised]-RC[reduced] 'product' state is compatible with rates of cyclic photosynthetic ET, at physiologically relevant light intensities.


Assuntos
Proteínas de Bactérias/metabolismo , Citocromos c/metabolismo , Luz , Fotossíntese , Rhodobacter sphaeroides/enzimologia , Proteínas de Bactérias/química , Citocromos c/química , Oxirredução
15.
Sci Rep ; 9(1): 10231, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308436

RESUMO

Phosphorus acquisition is critical for life. In low phosphate conditions, some species of bacteria have evolved mechanisms to import reduced phosphorus compounds, such as phosphite and hypophosphite, as alternative phosphorus sources. Uptake is facilitated by high-affinity periplasmic binding proteins (PBPs) that bind cargo in the periplasm and shuttle it to an ATP-binding cassette (ABC)-transporter in the bacterial inner membrane. PtxB and HtxB are the PBPs responsible for binding phosphite and hypophosphite, respectively. They recognize the P-H bond of phosphite/hypophosphite via a conserved P-H...π interaction, which confers nanomolar dissociation constants for their respective ligands. PtxB also has a low-level binding affinity for phosphate and hypophosphite, whilst HtxB can facilitate phosphite uptake in vivo. However, HtxB does not bind phosphate, thus the HtxBCDE transporter has recently been successfully exploited for biocontainment of genetically modified organisms by phosphite-dependent growth. Here we use a combination of X-ray crystallography, NMR and Microscale Thermophoresis to show that phosphite binding to HtxB depends on the protonation state of the ligand, suggesting that pH may effect the efficiency of phosphite uptake by HtxB in biotechnology applications.

16.
Biochim Biophys Acta Bioenerg ; 1860(7): 591-599, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247170

RESUMO

Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral cytochrome b6f (cytb6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytb6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytb6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 µs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytb6f are in opposite redox states, so complementary charges on the cytb6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytb6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the 'Z-scheme' of photosynthetic ET.


Assuntos
Complexo Citocromos b6f/metabolismo , Plastocianina/metabolismo , Análise de Célula Única/métodos , Spinacia oleracea/metabolismo , Complexo Citocromos b6f/química , Transporte de Elétrons , Oxirredução , Fotossíntese , Plastocianina/química , Ligação Proteica , Conformação Proteica
17.
Biochem J ; 476(13): 1875-1887, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31164400

RESUMO

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2--dependent insertion of a Mg2+ ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro, ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding (K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.


Assuntos
Liases/química , Magnésio/química , Proteínas Recombinantes/química , Synechocystis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Liases/genética , Domínios Proteicos , Proteínas Recombinantes/genética , Synechocystis/genética
18.
Plant Physiol ; 180(4): 2152-2166, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31186333

RESUMO

Grana stacking in plant chloroplast thylakoid membranes dynamically responds to the light environment. These dynamics have been linked to regulation of the relative antenna sizes of PSI and PSII (state transitions), the PSII repair cycle, and the regulation of photosynthetic electron transfer. Here, we used 3D structured illumination microscopy, a subdiffraction-resolution fluorescence imaging technique, to investigate the light-intensity dependence, kinetics, reversibility, and regulation of dynamic thylakoid stacking in spinach (Spinacia oleracea) and Arabidopsis (Arabidopsis thaliana). Low-intensity white light (150 µmol photons m-2 s-1) behaved similarly to light preferentially exciting PSII (660 nm), causing a reduction in grana diameter and an increased number of grana per chloroplast. By contrast, high-intensity white light (1000 µmol photons m-2 s-1), darkness, and light preferentially exciting PSI (730 nm) reversed these changes. These dynamics occurred with a half-time of 7 to 8 min and were accompanied by state transitions. Consistent with this, the dynamics were dependent on STN7 (light-harvesting complex II [LHCII] kinase) and TAP38 (LHCII phosphatase), which are required for state transitions but were unaffected by the absence of STN8 (PSII kinase) or PSII core phosphatase (PSII phosphatase). Unlike state transitions, however, thylakoid stacking dynamics did not rely on the presence of the LHCI and PSI subunit L phospho-LHCII binding sites on PSI. Since oligomerization of thylakoid curvature protein (CURT1A) was unaffected by the absence of STN7 or TAP38, we conclude that the primary determinant of dynamic thylakoid stacking is LHCII phosphorylation.

19.
Faraday Discuss ; 216(0): 57-71, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31016297

RESUMO

When light-harvesting complex II (LHCII), isolated from spinach, is adsorbed onto arrays of gold nanostructures formed by interferometric lithography, a pronounced splitting of the plasmon band is observed that is attributable to strong coupling of the localised surface plasmon resonance to excitons in the pigment-protein complex. The system is modelled as coupled harmonic oscillators, yielding an exciton energy of 2.24 ± 0.02 eV. Analysis of dispersion curves yields a Rabi energy of 0.25 eV. Extinction spectra of the strongly coupled system yield a resonance at 1.43 eV that varies as a function of the density of nanostructures in the array. The enhanced intensity of this feature is attributed to strong plasmon-exciton coupling. Comparison of data for a large number of light-harvesting complexes indicates that by control of the protein structure and/or pigment compliment it is possible to manipulate the strength of plasmon-exciton coupling. In strongly coupled systems, ultra-fast exchange of energy occurs between pigment molecules: coherent coupling between non-local excitons can be manipulated via selection of the protein structure enabling the observation of transitions that are not seen in the weak coupling regime. Synthetic biology thus provides a means to control quantum-optical interactions in the strong coupling regime.


Assuntos
Complexo de Proteína do Fotossistema II/química , Teoria Quântica , Biologia Sintética , Complexo de Proteína do Fotossistema II/metabolismo , Conformação Proteica
20.
J Phys Chem B ; 123(9): 2087-2093, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30739452

RESUMO

We used elastic incoherent neutron scattering (EINS) to find out if structural changes accompanying local hydrogen bond rupture are also reflected in global dynamical response of the protein complex. Chromatophore membranes from LH2-only strains of the photosynthetic bacterium Rhodobacter sphaeroides, with spheroidenone or neurosporene as the major carotenoids, were subjected to high hydrostatic pressure at ambient temperature. Optical spectroscopy conducted at high pressure confirmed rupture of tertiary structure hydrogen bonds. In parallel, we used EINS to follow average motions of the hydrogen atoms in LH2, which reflect the flexibility of this complex. A decrease of the average atomic mean square displacements of hydrogen atoms was observed up to a pressure of 5 kbar in both carotenoid samples due to general stiffening of protein structures, while at higher pressures a slight increase of the displacements was detected in the neurosporene mutant LH2 sample only. These data show a correlation between the local pressure-induced breakage of H-bonds, observed in optical spectra, with the altered protein dynamics monitored by EINS. The slightly higher compressibility of the neurosporene mutant sample shows that even subtle alterations of carotenoids are manifested on a larger scale and emphasize a close connection between the local structure and global dynamics of this membrane protein complex.

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