Paul D Barker

University of Cambridge, Cambridge, England, United Kingdom

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Publications (35)212.82 Total impact

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    ABSTRACT: Starch is a prominent component of the human diet and is hydrolysed by α-amylase post ingestion. Probing the mechanism of this process has proven challenging, due to the intrinsic heterogeneity of individual starch granules. By means of solution-state NMR, we demonstrate that flexible polysaccharide chains protruding from the solvent-exposed surfaces of waxy rice starch granules are highly mobile and that during hydrothermal treatment, when the granules swell, the number of flexible residues on the exposed surfaces increases by a factor of 15. Moreover, we show that these flexible chains are the primary substrates for α-amylase, being cleaved in the initial stages of hydrolysis. These findings allow us to conclude that the quantity of flexible α-glucan chains protruding from the granule surface will greatly influence the rate of energy acquisition from digestion of starch.
    Biomacromolecules 03/2015; 16(5). DOI:10.1021/acs.biomac.5b00190 · 5.75 Impact Factor
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    ABSTRACT: Previous theoretical studies of C3B have suggested that boron-doped graphite is a promising H2- and Li-storage material, with large maximum capacities. These characteristics could lead to exciting applications as a lightweight H2-storage material for automotive engines and as an anode in a new generation of batteries. However, for these applications to be realized a synthetic route to bulk C3B must be developed. Here we show the thermolysis of a single-source precursor (1,3-(BBr2)2C6H4) to produce graphitic C3B, thus allowing the characteristics of this elusive material to be tested for the first time. C3B was found to be compositionally uniform but turbostratically disordered. Contrary to theoretical expectations, the H2- and Li-storage capacities are lower than anticipated, results that can partially be explained by the disordered nature of the material. This work suggests that to model the properties of graphitic materials more realistically, the possibility of disorder must be considered.
    Angewandte Chemie 03/2015; 127(20). DOI:10.1002/ange.201412200
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    ABSTRACT: Previous theoretical studies of C3 B have suggested that boron-doped graphite is a promising H2 - and Li-storage material, with large maximum capacities. These characteristics could lead to exciting applications as a lightweight H2 -storage material for automotive engines and as an anode in a new generation of batteries. However, for these applications to be realized a synthetic route to bulk C3 B must be developed. Here we show the thermolysis of a single-source precursor (1,3-(BBr2 )2 C6 H4 ) to produce graphitic C3 B, thus allowing the characteristics of this elusive material to be tested for the first time. C3 B was found to be compositionally uniform but turbostratically disordered. Contrary to theoretical expectations, the H2 - and Li-storage capacities are lower than anticipated, results that can partially be explained by the disordered nature of the material. This work suggests that to model the properties of graphitic materials more realistically, the possibility of disorder must be considered. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Angewandte Chemie International Edition 03/2015; 54(20). DOI:10.1002/anie.201412200 · 11.26 Impact Factor
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    ABSTRACT: A series of organometallic complexes of the form [(PhH)Ru(amino acid)](+) have been synthesized using amino acids able to act as tridentate ligands. The straightforward syntheses gave enantiomerically pure complexes with two stereogenic centers due to the enantiopurity of the chelating ligands. Complexes were characterized in the solid-state and/or solution-state where the stability of the complex allowed. The propensity toward labilization of the coordinatively saturated complexes was investigated. The links between complex stability and structural features are very subtle. Nonetheless, H/D exchange rates of coordinated amino groups reveal more significant differences in reactivity linked to metallocycle ring size resulting in decreasing stability of the metallocycle as the amino acid side-chain length increases. The behavior of these systems in acid is unusual, apparently labilizing the carboxylate residue of the amino acid. This acid-catalyzed hemilability in an organometallic is relevant to the use of Ru(II) arenes in medicinal contexts due to the relatively low pH of cancerous cells.
    Inorganic Chemistry 03/2015; 54(7). DOI:10.1021/ic502051y · 4.79 Impact Factor
  • Tom G Scrase · Simon M Page · Paul D Barker · Sally R Boss
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    ABSTRACT: Under physiologically relevant conditions, cis-bis(2,2'-bipyridine)dichlororuthenium(ii), [cis-Ru(2,2'-bipy)2Cl2] was observed to bind to folic acid via replacement of the two chloride ligands. This binding was shown to be pH dependent and afforded diastereomers, the structures of which were determined by 1- and 2D NMR spectroscopic techniques. We propose that when studying the cytotoxicity of labile ruthenium complexes in cells, folate coordination should be considered.
    Dalton Transactions 04/2014; 43(22). DOI:10.1039/c4dt00081a · 4.20 Impact Factor
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    ABSTRACT: Decorative domains force amyloid fibers to adopt spiral ribbon morphologies, as opposed to the more common twisted ribbon. We model the effect of decorating domains as a perturbation to the relative orientation of β strands in a bilayered extended β-sheet. The model consists of minimal energy assemblies of rigid building blocks containing two anisotropic interacting ellipsoids. The relative orientation of the ellipsoids dictates the morphology of the resulting assembly. Amyloid structures derived from experiment are consistent with our model, and we use magnets to demonstrate that the frustration principle is scale and system independent. In contrast to other models of amyloid, our model isolates the effect of frustration from the fundamental interactions between building blocks to reveal the frustration rather than dependence of morphology on the physical interactions. Consequently, amyloid is viewed as a discrete molecular version of the more general macroscopic frustrated bilayer that is exemplified by Bauhinia seedpods. The model supports the idea that the interactions arising from an arbitrary peptide sequence can support an amyloid structure if a bilayer can form first, which suggests that supplementary protein sequences, such as chaperones or decorative domains, could play a significant role in stabilizing such bilayers and therefore in selecting morphology during nucleation. Our model provides a foundation for exploring the effects of frustration on higher-order superstructural polymorphic assemblies that may exhibit complex functional behavior. Two outstanding examples are the systematic kinking of decorated fibers and the nested frustration of the Bauhinia seedpod.
    The Journal of Physical Chemistry B 05/2013; 117(26). DOI:10.1021/jp4040503 · 3.30 Impact Factor
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    C J Forman · N Wang · Z Y Yang · C G Mowat · S Jarvis · C Durkan · P D Barker
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    ABSTRACT: Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.
    Nanotechnology 04/2013; 24(17):175102. DOI:10.1088/0957-4484/24/17/175102 · 3.67 Impact Factor
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    ABSTRACT: The construction of useful functional biomolecular components not currently part of the natural repertoire is central to synthetic biology. A new light-capturing ultra-high-efficiency energy transfer protein scaffold has been constructed by coupling the chromophore centers of two normally unrelated proteins: the autofluorescent protein enhanced green fluorescent protein (EGFP) and the heme-binding electron transfer protein cytochrome b(562) (cyt b(562)). Using a combinatorial domain insertion strategy, a variant was isolated in which resonance energy transfer from the donor EGFP to the acceptor cyt b(562) was close to 100% as evident by virtually full fluorescence quenching on heme binding. The fluorescence signal of the variant was also sensitive to the reactive oxygen species H(2)O(2), with high signal gain observed due to the release of heme. The structure of oxidized holoprotein, determined to 2.75 Å resolution, revealed that the two domains were arranged side-by-side in a V-shape conformation, generating an interchromophore distance of ~17 Å (14 Å edge-to-edge). Critical to domain arrangement is the formation of a molecular pivot point between the two domains as a result of different linker sequence lengths at each domain junction and formation of a predominantly polar interdomain interaction surface. The retrospective structural analysis has provided an explanation for the basis of the observed highly efficient energy transfer through chromophore arrangement in the directly evolved protein scaffold and provides an insight into the molecular principles by which to design new proteins with coupled functions.
    Journal of the American Chemical Society 07/2012; 134(33):13632-40. DOI:10.1021/ja301987h · 11.44 Impact Factor
  • Elizabeth B Sawyer · Paul D Barker
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    ABSTRACT: Cytochromes c covalently bind their heme prosthetic groups through thioether bonds between the vinyl groups of the heme and the thiols of a CXXCH motif within the protein. In Gram-negative bacteria, this process is catalyzed by the Ccm (cytochrome c maturation) proteins, also called System I. The Ccm proteins are found in the bacterial inner membrane, but some (CcmE, CcmG, CcmH, and CcmI) also have soluble functional domains on the periplasmic face of the membrane. Elucidation of the mechanisms involved in the transport and relay of heme and the apocytochrome from the bacterial cytosol into the periplasm, and their subsequent reaction, has proved challenging due to the fact that most of the proteins involved are membrane-associated, but recent progress in understanding some key components has thrown up some surprises. In this Review, we discuss advances in our understanding of this process arising from a substrate's point of view and from recent structural information about individual components.
    Protein & Cell 06/2012; 3(6):405-9. DOI:10.1007/s13238-012-2912-x · 2.85 Impact Factor
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    ABSTRACT: Self-assembled structures capable of mediating electron transfer are an attractive scientific and technological goal. Therefore, systematic variants of SH3-Cytochrome b(562) fusion proteins were designed to make amyloid fibers displaying heme-b(562) electron transfer complexes. TEM and AFM data show that fiber morphology responds systematically to placement of b(562) within the fusion proteins. UV-vis spectroscopy shows that, for the fusion proteins under test, only half the fiber-borne b(562) binds heme with high affinity. Cofactor binding also improves the AFM imaging properties and changes the fiber morphology through changes in cytochrome conformation. Systematic observations and measurements of fiber geometry suggest that longitudinal registry of subfilaments within the fiber, mediated by the interaction and conformation of the displayed proteins and their interaction with surfaces, gives rise to the observed morphologies, including defects and kinks. Of most interest is the role of small molecule modulation of fiber structure and mechanical stability. A minimum complexity model is proposed to capture and explain the fiber morphology in the light of these results. Understanding the complex interplay between these factors will enable a fiber design that supports longitudinal electron transfer.
    ACS Nano 02/2012; 6(2):1332-46. DOI:10.1021/nn204140a · 12.88 Impact Factor
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    ABSTRACT: An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.
    Journal of the American Chemical Society 08/2011; 133(36). · 11.44 Impact Factor
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    ABSTRACT: An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.
    Journal of the American Chemical Society 06/2011; 133(36):14160-3. DOI:10.1021/ja2017703 · 11.44 Impact Factor
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    ABSTRACT: The interaction of heme with the heme chaperone CcmE is central to our understanding of cytochrome c maturation, a complex post-translational process involving at least eight proteins in many Gram-negative bacteria and plant mitochondria. We have shown previously that Escherichia coli CcmE can interact with heme non-covalently in vitro, before forming a novel covalent histidine-heme bond, in a redox-sensitive manner. The function of CcmE is to bind heme in the periplasm before transferring it to apocytochromes c. In the absence of structural information on the complex of CcmE and heme, we have further characterized it by examining the binding of the soluble domain of CcmE (CcmE') to protoporphyrins containing metals other than Fe, namely Zn-, Sn-, Co- and Mn-protoporphyrin (PPIX). CcmE' demonstrated no affinity for the Zn- or Sn-containing protoporphyrins and low affinity for Mn(ii)-PPIX. High-affinity, reversible binding was, however, observed for Co(iii)-PPIX, which was highly sensitive to oxidation state as demonstrated by release of the ligand from the chaperone on reduction; no binding to Co(ii)-PPIX was observed. The non-covalent complex of CcmE' and Co(iii)-PPIX was characterized by non-denaturing mass spectrometry. The implications of these observations for the in vivo function of CcmE are discussed.
    Metallomics 01/2011; 3(4):363-8. DOI:10.1039/c0mt00085j · 3.98 Impact Factor
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    ABSTRACT: The system I cytochrome c maturation (Ccm) apparatus has been shown to handle a wide variety of apocytochrome substrates containing the CX(n)CH heme attachment sequence, where n = 2, 3, or 4 in natural sequences. When n = 5 or 6, the apparatus also appears to handle these substrates correctly, but close inspection reveals that the resulting mature cytochromes are mixtures of species containing extra mass. We have used accurate mass spectrometry to analyze peptide digests of matured Escherichia coli cytochrome cb(562) with n = 1, 5, or 6 and shown that an extra sulfur is sometimes incorporated into the heme-protein linkage. These unprecedented, aberrant persulfide linkages may shed new light upon the mechanism of the attachment of heme to substrate apocytochrome within the Ccm complex of E. coli.
    Journal of the American Chemical Society 03/2010; 132(14):4974-5. DOI:10.1021/ja908241v · 11.44 Impact Factor
  • Simon M Page · Sally R Boss · Paul D Barker
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    ABSTRACT: This review aims to bring the reader up to date with the more recent ruthenium compounds that have been synthesized and tested for their cytotoxicity. The chemistry of these transition metal complexes will be introduced and the basic principles that govern their common behavior outlined. The recent history of established compounds within this field will be presented alongside those that now represent the cutting-edge. The inherent variety within this class of compounds will lead the reader to appreciate their diversity and pose questions as to their similarities aside from the presence of a shared metal ion. This review aims to discuss and contextualize the state-of-the-art research within the context of the speculative advancement of this developing field. There is an evident need to specify the molecular and cellular targets of these drug molecules in order to ultimately elucidate their mode or modes of action. The evidence presented herein suggests that new avenues of research require novel analytical probes and methods for tracing the fate of ruthenium complexes in cells in order to understand their very promising cytotoxic activity.
    Future medicinal chemistry 06/2009; 1(3):541-59. DOI:10.4155/fmc.09.25 · 4.00 Impact Factor
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    ABSTRACT: c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys-Xaa-Xaa-Cys-His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b(562) containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b(562) to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CX(n)CH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.
    Biochemical Journal 04/2009; 419(1):177-84, 2 p following 184. DOI:10.1042/BJ20081999 · 4.78 Impact Factor
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    ABSTRACT: Telling fibrils: Diffusion data obtained by solution NMR spectroscopy from flexible regions of amyloid fibrils for both rotational and translation diffusion combined is of similar magnitude to that measured using AFM and TEM (see picture). Fibrils in solution are calculated to be somewhat longer on average than those deposited on surfaces for microscopy experiments, which can be partially attributed to the sensitivity of fibrils to fracture.
    Angewandte Chemie International Edition 04/2008; 47(18):3385-7. DOI:10.1002/anie.200703915 · 11.26 Impact Factor
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    ABSTRACT: NMR diffusion experiments employing pulsed field gradients are well established as sensitive probes of the displacement of individual nuclear spins in a sample. Conventionally such measurements are used as a measure of translational diffusion, but here we demonstrate that under certain conditions rotational motion will contribute very significantly to the experimental data. This situation occurs when at least one spatial dimension of the species under study exceeds the root mean square displacement associated with translational diffusion, and leads to anomalously large apparent diffusion coefficients when conventional analytical procedures are employed. We show that in such a situation the effective diffusion coefficient is a function of the duration of the diffusion delay used, and that this dependence provides a means of characterizing the dimensions of the species under investigation.
    The Journal of Chemical Physics 10/2007; 127(11):114505. DOI:10.1063/1.2759211 · 3.12 Impact Factor
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    ABSTRACT: Bacteria rely on their environment and/or host to acquire iron and have evolved specialized systems to sequester and transport heme. The heme uptake system HemRSTUV is common to proteobacteria, and a major challenge is to understand the molecular mechanism of heme binding and transfer between the protein molecules that underlie this heme transport relay process. In the Gram-negative pathogen Yersinia enterocolitica, the HemRSTUV system culminates with the cytoplasmic recipient HemS, which stores and delivers heme for cellular needs. HemS belongs to a family of proteins essential and unique to proteobacteria. Here we report on the binding mechanism of HemS based on structural data from its apo- and ligand-loaded forms. This heme carrier protein associates with its cargo through a novel, partly preformed binding pocket, formed between a large beta-sheet dome and a three-helix subdomain. In addition to a histidine interacting with the iron, the complex is stabilized by a distal non-coordinating arginine that packs along the porphyrin plane and extensive electrostatic contacts that firmly anchor the heme propionate groups within the protein. Comparison of apo- and ligand-bound HemS crystal structures reveals striking conformational changes that underlie a "heme-induced fit" binding mechanism. Local shifts in amino acid positions combine with global, rigid body-like domain movements, and together, these bring about a switch from an open, apo-form to a closed, bound state. This is the first report in which both liganded and unliganded forms of a heme transport protein are described, thus providing penetrating insights into its mechanism of heme binding and release.
    Journal of Biological Chemistry 11/2006; 281(43):32606-10. DOI:10.1074/jbc.M607516200 · 4.57 Impact Factor