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Benjamin T Goult,
Tom Zacharchenko,
Neil Bate,
Ricky Tsang,
Fiona Hey,
Alexandre R Gingras,
Paul R Elliott, Gordon C K Roberts,
Christoph Ballestrem,
David R Critchley,
Igor L Barsukov
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ABSTRACT: Talin activates integrins, couples them to F-actin and recruits vinculin to focal adhesions (FAs). Here we report the structural characterization of the talin rod, 13 helical bundles (R1-R13) organized into a compact cluster of 4-helix bundles (R2-R4) within a linear chain of 5-helix bundles. Nine of the bundles contain vinculin-binding sites (VBSs) - R2R3 are atypical each containing two VBSs. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding while RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs, and expression of the talin-binding domain of vinculin displaces RIAM from FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBSs, which recruit vinculin to stabilize the complex.
Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
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ABSTRACT: Conforming to convention: Evidence for compact and extended forms of cytochrome P450 reductase, believed to exist in dynamic equilibrium in solution, is found in the gas phase by using ion mobility spectrometry. The relative abundance of the two conformations can be influenced by the ionic strength of the solution from which they are electrosprayed.
Angewandte Chemie International Edition 06/2011; 50(36):8291-4. · 13.45 Impact Factor
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ABSTRACT: The potential of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium for biocatalysis and biotechnological application is widely acknowledged. The catalytic and structural analysis of the Ala82Phe mutant of P450 BM3 has shown that filling a hydrophobic pocket near the active site improved the binding of small molecules, such as indole (see Huang et al., J. Mol. Biol., 2007, 373, 633) and styrene. In this paper, additional mutations at Thr438 are shown to decrease the binding of and catalytic activity towards laurate, whereas they significantly increased the stereo-specificity of styrene epoxidation. Production of R-styrene oxide with 48% and 64% e.e., respectively, was achieved by the Ala82Phe-Thr438Leu and Ala82Phe-Thr438Phe mutants. These structure-based mutants of P450 BM3 illustrate the promise of rational design of synthetically useful biocatalysts for regio- and stereo- specific mono-oxygenation reactions.
Metallomics 01/2011; 3(4):410-6. · 3.90 Impact Factor
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ABSTRACT: FERM domains are found in a diverse superfamily of signaling and adaptor proteins at membrane interfaces. They typically consist of three separately folded domains (F1, F2, F3) in a compact cloverleaf structure. The crystal structure of the N-terminal head of the integrin-associated cytoskeletal protein talin reported here reveals a novel FERM domain with a linear domain arrangement, plus an additional domain F0 packed against F1. While F3 binds β-integrin tails, basic residues in F1 and F2 are required for membrane association and for integrin activation. We show that these same residues are also required for cell spreading and focal adhesion assembly in cells. We suggest that the extended conformation of the talin head allows simultaneous binding to integrins via F3 and to PtdIns(4,5)P2-enriched microdomains via basic residues distributed along one surface of the talin head, and that these multiple interactions are required to stabilize integrins in the activated state.
Structure 10/2010; 18(10):1289-99. · 6.35 Impact Factor
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ABSTRACT: Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359-1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.
Journal of Biological Chemistry 09/2010; 285(38):29577-87. · 4.77 Impact Factor
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ABSTRACT: Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains
an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However,
determining the structure of the central part of the rod has proved problematic. Residues 1359–1659 are homologous to the
MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential
helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles
forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle
contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the
4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than
other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic,
whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding.
Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.
Journal of Biological Chemistry 09/2010; 285(38):29577-29587. · 4.77 Impact Factor
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ABSTRACT: Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815-1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843-1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction.
FEBS letters 06/2010; 584(11):2237-41. · 3.54 Impact Factor
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Benjamin T Goult,
Mohamed Bouaouina,
Paul R Elliott,
Neil Bate,
Bipin Patel,
Alexandre R Gingras,
J Günter Grossmann, Gordon C K Roberts,
David A Calderwood,
David R Critchley,
Igor L Barsukov
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ABSTRACT: Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for beta-integrin tails, F0 and F1 are also required for activation of beta1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of beta1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.
The EMBO Journal 02/2010; 29(6):1069-80. · 9.20 Impact Factor
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ABSTRACT: NADPH-cytochrome P450 reductase (CPR), a diflavin reductase, plays a key role in the mammalian P450 mono-oxygenase system.
In its crystal structure, the two flavins are close together, positioned for interflavin electron transfer but not for electron
transfer to cytochrome P450. A number of lines of evidence suggest that domain motion is important in the action of the enzyme.
We report NMR and small-angle x-ray scattering experiments addressing directly the question of domain organization in human
CPR. Comparison of the 1H-15N heteronuclear single quantum correlation spectrum of CPR with that of the isolated FMN domain permitted identification of
residues in the FMN domain whose environment differs in the two situations. These include several residues that are solvent-exposed
in the CPR crystal structure, indicating the existence of a second conformation in which the FMN domain is involved in a different
interdomain interface. Small-angle x-ray scattering experiments showed that oxidized and NADPH-reduced CPRs have different
overall shapes. The scattering curve of the reduced enzyme can be adequately explained by the crystal structure, whereas analysis
of the data for the oxidized enzyme indicates that it exists as a mixture of approximately equal amounts of two conformations,
one consistent with the crystal structure and one a more extended structure consistent with that inferred from the NMR data.
The correlation between the effects of adenosine 2′,5′-bisphosphate and NADPH on the scattering curve and their effects on
the rate of interflavin electron transfer suggests that this conformational equilibrium is physiologically relevant.
Journal of Biological Chemistry 12/2009; 284(52):36628-36637. · 4.77 Impact Factor
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ABSTRACT: NADPH-cytochrome P450 reductase (CPR), a diflavin reductase, plays a key role in the mammalian P450 mono-oxygenase system. In its crystal structure, the two flavins are close together, positioned for interflavin electron transfer but not for electron transfer to cytochrome P450. A number of lines of evidence suggest that domain motion is important in the action of the enzyme. We report NMR and small-angle x-ray scattering experiments addressing directly the question of domain organization in human CPR. Comparison of the (1)H-(15)N heteronuclear single quantum correlation spectrum of CPR with that of the isolated FMN domain permitted identification of residues in the FMN domain whose environment differs in the two situations. These include several residues that are solvent-exposed in the CPR crystal structure, indicating the existence of a second conformation in which the FMN domain is involved in a different interdomain interface. Small-angle x-ray scattering experiments showed that oxidized and NADPH-reduced CPRs have different overall shapes. The scattering curve of the reduced enzyme can be adequately explained by the crystal structure, whereas analysis of the data for the oxidized enzyme indicates that it exists as a mixture of approximately equal amounts of two conformations, one consistent with the crystal structure and one a more extended structure consistent with that inferred from the NMR data. The correlation between the effects of adenosine 2',5'-bisphosphate and NADPH on the scattering curve and their effects on the rate of interflavin electron transfer suggests that this conformational equilibrium is physiologically relevant.
Journal of Biological Chemistry 10/2009; 284(52):36628-37. · 4.77 Impact Factor
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ABSTRACT: Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the beta-lactamase II from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K(1)/K(2)> or =5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K(2)<80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its alpha-helical content, presumably associated with enhanced flexibility.
Journal of Molecular Biology 09/2009; 392(5):1278-91. · 4.00 Impact Factor
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ABSTRACT: Talin is a large cytoskeletal protein (2541 amino acid residues) which plays a key role in integrin-mediated events that are crucial for cell adhesion, migration, proliferation and survival. This review summarises recent work on the structure of talin and on some of the structurally better defined interactions with other proteins. The N-terminal talin head (approx. 50 kDa) consists of an atypical FERM domain linked to a long flexible rod (approx. 220 kDa) made up of a series of amphipathic helical bundle domains. The F3 FERM subdomain in the head binds the cytoplasmic tail of integrins, but this interaction can be inhibited by an interaction of F3 with a helical bundle in the talin rod, the so-called "autoinhibited form" of the molecule. The talin rod contains a second integrin-binding site, at least two actin-binding sites and a large number of binding sites for vinculin, which is important in reinforcing the initial integrin-actin link mediated by talin. The vinculin binding sites are defined by hydrophobic residues buried within helical bundles, and these must unfold to allow vinculin binding. Recent experiments suggest that this unfolding may be mediated by mechanical force exerted on the talin molecule by actomyosin contraction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12551-009-0009-4) contains supplementary material, which is available to authorized users.
Biophysical Reviews 08/2009; 1(2):61-69.
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ABSTRACT: Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655-1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316-326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with beta3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.
Journal of Biological Chemistry 04/2009; 284(22):15097-106. · 4.77 Impact Factor
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Alexandre R Gingras,
Wolfgang H Ziegler,
Andrey A Bobkov,
M Gordon Joyce,
Domenico Fasci,
Mirko Himmel,
Sven Rothemund,
Anett Ritter,
J Günter Grossmann,
Bipin Patel,
Neil Bate,
Benjamin T Goult,
Jonas Emsley,
Igor L Barsukov, Gordon C K Roberts,
Robert C Liddington,
Mark H Ginsberg,
David R Critchley
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ABSTRACT: The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin beta-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, "IBS2-A" (1974-2139) and "IBS2-B" (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.
Journal of Biological Chemistry 02/2009; 284(13):8866-76. · 4.77 Impact Factor
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ABSTRACT: Cytochrome P450 3A4, a major drug-metabolizing enzyme in man, is well known to show non-Michaelis-Menten steady-state kinetics for a number of substrates, indicating that more than one substrate can bind to the enzyme simultaneously, but it has proved difficult to obtain reliable estimates of exactly how many substrate molecules can bind. We have used a simple method involving studies of the effect of large inhibitors on the Hill coefficient to provide improved estimates of substrate stoichiometry from simple steady-state kinetics. Using a panel of eight inhibitors, we show that at least four molecules of the widely used CYP3A4 substrate 7-benzyloxyquinoline can bind simultaneously to the enzyme. Computational docking studies show that this is consistent with the recently reported crystal structures of the enzyme. In the case of midazolam, which shows simple Michaelis-Menten kinetics, the inhibitor effects demonstrate that two molecules must bind simultaneously, consistent with earlier evidence, whereas for diltiazem, the experiments provide no evidence for the binding of more than one molecule. The consequences of this "inhibitor-induced cooperativity" for the prediction of pharmacokinetics and drug-drug interactions are discussed.
Drug metabolism and disposition: the biological fate of chemicals 10/2008; 36(10):2136-44. · 3.74 Impact Factor
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ABSTRACT: Talin is a large cytoskeletal protein that is involved in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton, colocalising with the integrins in focal adhesions (FAs). However, at the leading edge of motile cells, talin colocalises with the hyaluronan receptor layilin in what are thought to be transient adhesions, some of which subsequently mature into more stable FAs. During this maturation process, layilin is replaced with integrins, which are highly clustered in FAs, where localised production of PI(4,5)P(2) by type 1 phosphatidyl inositol phosphate kinase type 1gamma (PIPK1gamma) is thought to play a role in FA assembly. The talin FERM F3 subdomain binds both the integrin beta-subunit cytoplasmic domain and PIPK1gamma, and these interactions are understood in detail at the atomic level. The talin F3 domain also binds to short sequences in the layilin cytoplasmic domain, and here we report the structure of the talin/layilin complex, which shows that talin binds integrins, PIPK1gamma and layilin in similar although subtly different ways. Based on structure comparisons, we designed a set of talin F3 mutations that selectively affected the affinity of talin for its targets, as determined by stopped-flow fluorescence measurements. Such mutations will help to assess the importance of the interactions between talin and its various ligands in cell adhesion and migration.
Journal of Molecular Biology 09/2008; 382(1):112-26. · 4.00 Impact Factor
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Benoît M R Liénard,
Gianpiero Garau,
Louise Horsfall,
Andreas I Karsisiotis,
Christian Damblon,
Patricia Lassaux,
Cyril Papamicael, Gordon C K Roberts,
Moreno Galleni,
Otto Dideberg,
Jean-Marie Frère,
Christopher J Schofield
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ABSTRACT: The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible.
Organic & Biomolecular Chemistry 08/2008; 6(13):2282-94. · 3.70 Impact Factor
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ABSTRACT: Talin is a large dimeric 270 kDa adapter protein which binds the cytoplasmic face of a subset of integrin beta-subunits and couples them to the actin cytoskeleton. Here we report the near complete 15N, 13C and 1H chemical shift assignments for the C-terminal actin-binding domain.
Biomolecular NMR Assignments 07/2008; 2(1):17-9. · 0.72 Impact Factor
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Alexandre R Gingras,
Neil Bate,
Benjamin T Goult,
Larnele Hazelwood,
Ilona Canestrelli,
J Günter Grossmann,
HongJun Liu,
Nicholas S M Putz, Gordon C K Roberts,
Niels Volkmann,
Dorit Hanein,
Igor L Barsukov,
David R Critchley
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ABSTRACT: Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.
The EMBO Journal 02/2008; 27(2):458-69. · 9.20 Impact Factor
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Benoît M. R. Liénard,
Gianpiero Garau,
Louise Horsfall,
Andreas I. Karsisiotis,
Christian Damblon,
Patricia Lassaux,
Cyril Papamicael, Gordon C. K. Roberts,
Moreno Galleni,
Otto Dideberg,
Jean-Marie Frère,
Christopher J. Schofield
Organic & Biomolecular Chemistry - ORG BIOMOL CHEM. 01/2008; 6(13).
