C Neil Hunter

The University of Sheffield, Sheffield, England, United Kingdom

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Publications (257)1229.04 Total impact

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    ABSTRACT: The photocatalytic self-cleaning characteristics of titania facilitate the fabrication of re-useable templates for protein nanopatterning. Titania nanostructures were fabricated over square centimeter areas by interferometric lithography (IL) and nanoimprint lithography (NIL). Using a Lloyd's mirror two-beam interferometer, self-assembled monolayers of alkylphosphonates adsorbed on the native oxide of a Ti film were patterned by photocatalytic nanolithography. In regions exposed to a maximum in the interferogram, the monolayer was removed by photocatalytic oxidation. In regions exposed to an intensity minimum, the monolayer remained intact. After exposure, the sample was etched in piranha solution to yield Ti nanostructures with widths as small as 30 nm. NIL was performed by using a silicon stamp to imprint a spin-cast film of titanium dioxide resin; after calcination and reactive ion etching, TiO2 nanopillars were formed. For both fabrication techniques, subsequent adsorption of an oligo(ethylene glycol) functionalized trichlorosilane yielded an entirely passive, protein-resistant surface. Near-UV exposure caused removal of this protein-resistant film from the titania regions by photocatalytic degradation, leaving the passivating silane film intact on the silicon dioxide regions. Proteins labeled with fluorescent dyes were adsorbed to the titanium dioxide regions, yielding nanopatterns with bright fluorescence. Subsequent near-UV irradiation of the samples removed the protein from the titanium dioxide nanostructures by photocatalytic degradation facilitating the adsorption of a different protein. The process was repeated multiple times. These simple methods appear to yield durable, re-useable samples that may be of value to laboratories that require nanostructured biological interfaces but do not have access to the infrastructure required for nanofabrication.
    ACS Nano 06/2015; DOI:10.1021/acsnano.5b01636
  • Interface focus: a theme supplement of Journal of the Royal Society interface 05/2015; 5(4):20150005-20150005. DOI:10.1098/rsfs.2015.0005
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    ABSTRACT: Light-harvesting 2 complexes (LH2) from a genetically modified strain of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. Carotenoid synthesis in the Rba. sphaeroides strain was engineered to redirect carotenoid production away from spheroidene into the spirilloxanthin synthesis pathway. The strain assembles LH2 antennas with substantial amounts of spirilloxanthin (total double-bond conjugation length N = 13) if grown anaerobically and of keto-bearing long-chain analogs [2-ketoanhydrorhodovibrin (N = 13), 2-ketospirilloxanthin (N = 14) and 2,2'-diketospirilloxanthin (N = 15)] if grown semi-aerobically (with ratios that depend on growth conditions). We present the photophysical, electronic, and vibrational properties of these carotenoids, both isolated in organic media and assembled within LH2 complexes. Measurements of excited-state energy transfer to the array of excitonically coupled bacteriochlorophyll a molecules (B850) show that the mean lifetime of the first singlet excited state (S1) of the long-chain (N ≥ 13) carotenoids does not change appreciably between organic media and the protein environment. In each case, the S1 state appears to lie lower in energy than that of B850. The energy-transfer yield is ~0.4 in LH2 (from the strain grown aerobically or semi-aerobically), which is less than half that achieved for LH2 that contains short-chain (N ≤ 11) analogues. Collectively, the results suggest that the S1 excited state of the long-chain (N ≥ 13) carotenoids participates little if at all in carotenoid-to-BChl a energy transfer, which occurs predominantly via the carotenoid S2 excited state in these antennas. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 04/2015; 374(6-7). DOI:10.1016/j.bbabio.2015.04.001
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    ABSTRACT: Stark absorption spectroscopy was applied to clarify the structural differences between carotenoids bound to the B800-820 and B800-850 LH2 complexes from a purple photosynthetic bacterium Phaeospirillum (Phs.) molischianum DSM120. The former complex is produced when the bacteria are grown under stressed conditions of low temperature and dim light. These two LH2 complexes bind carotenoids with similar composition, 10% lycopene and 80% rhodopin, each with the same number of conjugated C=C double bonds (n = 11). Quantitative classical and semi-quantum chemical analyses of Stark absorption spectra recorded in the carotenoid absorption region reveal that the absolute values of the difference dipole moments |Δμ| have substantial differences (2 [D/f]) for carotenoids bound to either B800-820 or B800-850 complexes. The origin of this striking difference in the |Δμ| values was analyzed using the X-ray crystal structure of the B800-850 LH2 complex from Phs. molischianum DSM119. Semi-empirical molecular orbital calculations predict structural deformations of the major carotenoid, rhodopin, bound within the B800-820 complex. We propose that simultaneous rotations around neighboring C=C and C-C bonds account for the differences in the 2 [D/f] of the |Δμ| value. The plausible position of the rotation is postulated to be located around C21-C24 bonds of rhodopin. Copyright © 2014. Published by Elsevier Inc.
    Archives of Biochemistry and Biophysics 12/2014; 572. DOI:10.1016/j.abb.2014.12.015
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    ABSTRACT: Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal 'head' region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH-I-D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain.
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    ABSTRACT: Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated CC bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis. Copyright © 2014. Published by Elsevier B.V.
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 10/2014; 1847(2):189-201. DOI:10.1016/j.bbabio.2014.10.004
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    Daniel P. Canniffe, C. Neil Hunter
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    ABSTRACT: Bacteriochlorophyll b has the most red-shifted absorbance maximum of all naturally-occurring photopigments. It has a characteristic ethylidene group at the C8 position in place of the more common ethyl group, the product of a C8-vinyl reductase, which is carried by the majority of chlorophylls and bacteriochlorophylls used in photosynthesis. The subsequent and first step exclusive to bacteriochlorophyll biosynthesis, the reduction of the C7 = C8 bond, is catalyzed by chlorophyllide oxidoreductase. It has been demonstrated that the enzyme from bacteriochlorophyll a-utilizing bacteria can catalyze the formation of compounds carrying an ethyl group at C8 from both ethyl- and vinyl-carrying substrates, indicating a surprising additional C8-vinyl reductase function, while the enzyme from organisms producing BChl b could only catalyze C7 = C8 reduction with a vinyl substrate, but this product carried an ethylidene group at the C8 position. We have replaced the native chlorophyllide oxidoreductase-encoding genes of Rhodobacter sphaeroides with those from Blastochloris viridis, but the switch from bacteriochlorophyll a to b biosynthesis is only detected when the native conventional C8-vinyl reductase is absent. We propose a non-enzymatic mechanism for ethylidene group formation based on the absence of cellular C8-vinyl reductase activity.
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 10/2014; 1837(10). DOI:10.1016/j.bbabio.2014.07.011
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    ABSTRACT: In the purple phototrophic bacterium Rhodobacter sphaeroides many protein complexes congregate within the membrane to form operational photosynthetic units consisting of arrays of light-harvesting LH2 complexes and monomeric and dimeric reaction center-light-harvesting 1-PufX (RC-LH1-PufX) &'core&' complexes. Each half of a dimer complex consists of a RC surrounded by 14 LH1 αβ subunits, with two BChls sandwiched between each αβ pair of transmembrane helices. We used atomic force microscopy (AFM) to investigate the assembly of single molecules of the RC-LH1-PufX complex using membranes prepared from LH2-minus mutants. When the RC and PufX components were also absent AFM revealed a series of LH1 variants where the repeating α1β1(Bchl)2 units had formed rings of variable size, ellipses, and spirals and also arcsthat could be assembly products. The spiral complexes occur when the LH1 ring has failed to close, and short arcs are suggestive of prematurely terminated LH1 complex assembly. In the absence of RCs we occasionally observed captive proteins enclosed by the LH1 ring. When production of LH1 units was restricted by lowering the relative levels of the cognate pufBA transcript, we imaged a mixture of complete RC-LH1 core complexes, empty LH1 rings and isolated RCs, leading us to conclude that once a RC associates with the first α1β1(Bchl)2 subunit, co-operative associations between subsequent subunits and the RC tend to drive LH1 ring assembly to completion.
    Journal of Biological Chemistry 09/2014; 289(43). DOI:10.1074/jbc.M114.596585
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    ABSTRACT: The cytochrome b6f (cytb6f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytb6f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytb6f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytb6f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytb6f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytb6f complexes. We suggest that the close proximity between PSII and cytb6f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.
    The Plant Cell 07/2014; 26(7). DOI:10.1105/tpc.114.127233
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    ABSTRACT: We describe a fast, simple method for the fabrication of re-usable, robust gold nanostructures over macroscopic (cm2) areas. A wide range of nanostructure morphologies is accessible in a combinatorial fashion. Self-assembled monolayers (SAMs) of alkylthiolates on chromium-primed polycrystalline gold films are patterned using a Lloyd's mirror interferometer and etched using mercaptoethylamine in ethanol in a rapid process that does not require access to clean-room facilities. The use of a Cr adhesion layer facilitates the cleaning of specimens by immersion in piranha solution, enabling their repeated re-use without significant change in their absorbance spectra over two years. A library of 200 different nanostructures was prepared, and found to exhibit a range of optical behavior. Annealing yielded structures with a uniformly high degree of crystallinity that exhibited strong plasmon bands. Using a combinatorial approach, correlations were established between the pre-annealing morphologies (determined by the fabrication conditions) and the post-annealing optical properties that enabled specimens to be prepared "to order" with a selected localized surface plasmon resonance. The refractive index sensitivity of gold nanostructures formed in this way was found to correlate closely with measurements reported for structures fabricated by other methods. Strong enhancements were observed in the Raman spectra of tetra-tert-butyl unsubstituted phthalocyanine (TTB-H2Pc). The shift in the position of the plasmon band after site-specific attachment of histidine-tagged green fluorescent protein (His-GFP) and bacteriochlorophyll a was measured for a range of nanostructured films, enabling the rapid identification of the one that yielded the largest shift. This approach offers a simple route to the production of durable, reusable, macroscopic arrays of gold nanostructures with precisely controllable morphologies.
    ACS Nano 07/2014; 8(8). DOI:10.1021/nn5014319
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    ABSTRACT: A simple and robust nanolithographic method, that allows sub-100 nm chemical patterning on a range of oxide surfaces, was developed in order to fabricate nanoarrays of plant light harvesting LHCII complexes. The site-specific immobilization and the preserved functionality of the LHCII complexes were confirmed by fluorescence emission spectroscopy. Nanopatterned LHCII trimers could be reversibly switched between fluorescent and quenched states by controlling the detergent concentration in the imaging buffer. A three-fold quenching of the average fluorescence intensity was accompanied by a decrease in the average (amplitude weighted) fluorescence lifetime from approximately 2.24 ns to approximately 0.4 ns, attributed to the intrinsic ability of LHCII to switch between fluorescent and quenched states upon changes in its conformational state. The nanopatterning methodology was extended by immobilizing a second protein, the enhanced Green Fluorescent Protein (EGFP), onto LHCII-free areas of the chemically patterned surfaces. This very simple surface chemistry, which allows simultaneous selective immobilization and therefore sorting of the two types of protein molecules on the surface, is a key underpinning step towards the integration of LHCII into switchable biohybrid antenna constructs.
    Langmuir 07/2014; 30(28). DOI:10.1021/la501483s
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    ABSTRACT: Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.
    Biochimica et Biophysica Acta 07/2014; 1837(10). DOI:10.1016/j.bbabio.2014.02.003
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e122. DOI:10.1016/j.bbabio.2014.05.326
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    ABSTRACT: In this study, we use the photosynthetic purple bacterium Rhodobacter sphaeroides to find out how the acclimation of photosynthetic apparatus to growth conditions influence the rates of energy migration towards the reaction center traps and the efficiency of charge separation at the reaction centers. To answer these questions we measured the spectral and picosecond kinetic fluorescence responses as a function of excitation intensity in membranes prepared from cells grown under different illumination conditions. A kinetic model analysis yielded the microscopic rate constants that characterize the energy transfer and trapping inside the photosynthetic unit as well as the dependence of exciton trapping efficiency on the ratio of the peripheral LH2 and core LH1 antenna complexes, and on the wavelength of the excitation light. A high quantum efficiency of trapping over 80% was observed in most cases, which decreased toward shorter excitation wavelengths within the near infrared absorption band. At a fixed excitation wavelength the efficiency declines with the LH2/LH1 ratio. From the perspective of the ecological habitat of the bacteria the higher population of peripheral antenna facilitates growth under dim light even though the energy trapping is slower in low light adapted membranes. The similar values for the trapping efficiencies in all samples imply a robust photosynthetic apparatus that functions effectively at a variety of light intensities.
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 06/2014; 1837(10). DOI:10.1016/j.bbabio.2014.06.007
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    ABSTRACT: Most of the chlorophylls and bacteriochlorophylls utilised for light harvesting by phototrophic organisms carry an ethyl group at the C8 position of the molecule, the product of a C8-vinyl reductase acting on a chlorophyll/bacteriochlorophyll biosynthetic precursor. Two unrelated classes of C8-vinyl reductase are known to exist, BciA and BciB, found in the purple phototroph Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp. PCC6803, respectively. We constructed strains of each bacterium with the native C8-vinyl reductase swapped for the other class of the enzyme, and combined these replacements with a series of deletions of the native bch and chl genes. In vivo data indicate that the preferred substrates for both classes of the enzyme is C8-vinyl chlorophyllide, with C8-vinyl protochlorophyllide reduced only under conditions in which this pigment accumulates as a result of perturbed formation of chlorophyllide.
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    ABSTRACT: A new cysteine-based methacrylic monomer (CysMA) was conveniently synthesized via selective thia-Michael addition of a commercially-available methacrylate-acrylate precursor in aqueous solution without recourse to protecting group chemistry. Poly(cysteine methacrylate) (PCysMA) brushes were grown from the surface of silicon wafers by atom-transfer radical polymerization. Brush thicknesses of ca. 27 nm were achieved within 270 min at 20 oC. Each CysMA residue comprises a primary amine and a carboxylic acid. Surface zeta potential and atomic force microscopy (AFM) studies of the pH-responsive PCysMA brushes confirm that they are highly extended either below pH 2 or above pH 9.5, since they possess either cationic or anionic character, respectively. At intermediate pH, PCysMA brushes are zwitterionic. At physiological pH, they exhibit excellent resistance to biofouling and negligible cytotoxicity. PCysMA brushes undergo photodegradation: AFM topographical imaging indicates significant mass loss from the brush layer, while XPS studies confirm that exposure to UV radiation produces surface aldehyde sites that can be subsequently derivatized with amines. UV exposure using a photomask yielded sharp, well-defined micro-patterned PCysMA brushes functionalized with aldehyde groups that enable conjugation to green fluorescent protein (GFP). Nano-patterned PCysMA brushes were obtained using interference lithography and confocal microscopy again confirmed the selective conjugation of GFP. Finally, PCysMA undergoes complex base-catalyzed degradation in alkaline solution, leading to the elimination of several small molecules. However, good long-term chemical stability was observed when PCysMA brushes were immersed in aqueous solution at physiological pH.
    Journal of the American Chemical Society 06/2014; 136(26). DOI:10.1021/ja503400r
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    ABSTRACT: The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy(P2A) complexes may have attached to the TatAyCy complex. EM and AFM analysis shows that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9-16 nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500 nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10 - 12 nm comparable to the size of single TatAy(P2A) complexes. The combined data indicate that the mutation leads to super-assembly of TatAy(P2A) complexes and we propose that an individual TatAy(P2A) complex assembles initially with a TatAy(P2A)Cy complex, after which further TatAy(P2A) complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.
    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 05/2014; 1843(9). DOI:10.1016/j.bbamcr.2014.05.009
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    ABSTRACT: Macromolecular membrane assemblies of chlorophyll-protein complexes efficiently harvest and trap light energy for photosynthesis. To investigate the delivery of chlorophylls to the newly synthesized photosystem apoproteins, a terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), was tagged in the cyanobacterium Synechocystis PCC 6803 (Synechocystis) and used as bait in pull-down experiments. We retrieved an enzymatically active complex comprising ChlG and the high-light-inducible protein HliD, which associates with the Ycf39 protein, a putative assembly factor for photosystem II, and with the YidC/Alb3 insertase. 2D electrophoresis and immunoblotting also provided evidence for the presence of SecY and ribosome subunits. The isolated complex contained chlorophyll, chlorophyllide, and carotenoid pigments. Deletion of hliD elevated the level of the ChlG substrate, chlorophyllide, more than 6-fold; HliD is apparently required for assembly of FLAG-ChlG into larger complexes with other proteins such as Ycf39. These data reveal a link between chlorophyll biosynthesis and the Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. We expect that this close physical linkage coordinates the arrival of pigments and nascent apoproteins to produce photosynthetic pigment-protein complexes with minimal risk of accumulating phototoxic unbound chlorophylls.
    The Plant Cell 03/2014; 26(3). DOI:10.1105/tpc.114.124495
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    ABSTRACT: The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAyP2A complexes may have attached to the TatAyCy complex. EM and AFM analysis shows that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9-16 nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500 nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10 – 12 nm comparable to the size of single TatAyP2A complexes. The combined data indicate that the mutation leads to super-assembly of TatAyP2A complexes and we propose that an individual TatAyP2A complex assembles initially with a TatAyP2ACy complex, after which further TatAyP2A complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.
    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 01/2014;

Publication Stats

8k Citations
1,229.04 Total Impact Points

Institutions

  • 1989–2015
    • The University of Sheffield
      • • Department of Molecular Biology and Biotechnology
      • • Department of Chemistry
      • • Centre for Photosynthesis Research (Robert Hill Institute)
      Sheffield, England, United Kingdom
  • 2012
    • University of Tartu
      • Institute of Molecular and Cell Biology
      Dorpat, Tartu, Estonia
  • 2010
    • University of Illinois, Urbana-Champaign
      • Beckman Institute for Advanced Science and Technology
      Urbana, IL, United States
  • 1998–2009
    • VU University Amsterdam
      • • Department of Physics and Astronomy
      • • Division of Theoretical Chemistry
      Amsterdam, North Holland, Netherlands
  • 1980–2008
    • Rutgers, The State University of New Jersey
      • • Department of Chemical Biology
      • • Department of Molecular Biology and Biochemistry
      New Brunswick, NJ, United States
  • 2006
    • Jagiellonian University
      Cracovia, Lesser Poland Voivodeship, Poland
  • 2004
    • Universiteit Twente
      Enschede, Overijssel, Netherlands
  • 1996–2002
    • Lund University
      • Department of Physical Chemistry
      Lund, Skane, Sweden
  • 2000
    • University of Glasgow
      Glasgow, Scotland, United Kingdom
  • 1997–1998
    • Northwestern University
      • Department of Cell and Molecular Biology
      Evanston, Illinois, United States
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
    • Stanford University
      • Department of Chemistry
      Palo Alto, California, United States
  • 1995
    • University of Amsterdam
      • Department of Physics and Astronomy
      Amsterdam, North Holland, Netherlands
  • 1989–1993
    • Umeå University
      Umeå, Västerbotten, Sweden
  • 1988–1993
    • Imperial Valley College
      • Department of Pure and Applied Biology
      Imperial, California, United States
  • 1986–1989
    • Imperial College London
      Londinium, England, United Kingdom
  • 1979–1985
    • University of Bristol
      • Medical School
      Bristol, England, United Kingdom