Dean A A Myles

Oak Ridge National Laboratory, Oak Ridge, Florida, United States

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Publications (83)329.91 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Many bacteria exist in a state of feast or famine where high nutrient availability leads to periods of growth followed by nutrient scarcity and growth stagnation. To adapt to the constantly changing nutrient flux, metabolite acquisition systems must be able to function over a broad range. This however creates difficulties as nutrient concentrations vary over many orders of magnitude, requiring metabolite acquisition systems to simultaneously balance ligand specificity and the dynamic range in which a response to a metabolite is elicited. Here we present how a gene duplication of a periplasmic binding protein (PBP) in a mannose ABC transport system potentially resolves this dilemma through gene functionalization. Determination of ligand binding affinities and specificities of the gene duplicates with fluorescence and circular dichroism demonstrate that although the binding specificity is maintained the Kd values for the same ligand differ over three orders of magnitude. These results suggest this metabolite acquisition system can transport ligand at both low and high environmental concentrations while preventing saturation with related and less preferentially metabolized compounds. The X-ray crystal structures of the beta mannose bound proteins help clarify the structural basis of gene functionalization and reveal that affinity and specificity are potentially encoded in different regions of the binding site. These studies suggest a possible functional role and adaptive advantage for the presence of two PBPs in ABC transport systems and a way bacteria can adapt to varying nutrient flux through functionalization of gene duplicates.
    The Journal of biological chemistry. 09/2014;
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    ABSTRACT: Small-angle neutron scattering (SANS) is a powerful tool for characterizing complex disordered materials, including biological materials. The Bio-SANS instrument of the High Flux Isotope Reactor of Oak Ridge National Laboratory (ORNL) is a high-flux low-background SANS instrument that is, uniquely among SANS instruments, dedicated to serving the needs of the structural biology and biomaterials communities as an open-access user facility. Here, the technical specifications and performance of the Bio-SANS are presented. Sample environments developed to address the needs of the user program of the instrument are also presented. Further, the isotopic labeling and sample preparation capabilities available in the Bio-Deuteration Laboratory for users of the Bio-SANS and other neutron scattering instruments at ORNL are described. Finally, a brief survey of research performed using the Bio-SANS is presented, which demonstrates the breadth of the research that the instrument's user community engages in.
    Journal of Applied Crystallography 08/2014; 47(4). · 3.34 Impact Factor
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    ABSTRACT: Cel7A (previously known as cellobiohydrolase I) from Hypocrea jecorina was crystallized in two crystalline forms, neither of which have been previously reported. Both forms co-crystallize under the same crystallization conditions. The first crystal form belonged to space group C2, with unit-cell parameters a = 152.5, b = 44.9, c = 57.6 Å, β = 101.2°, and diffracted X-rays to 1.5 Å resolution. The second crystal form belonged to space group P6322, with unit-cell parameters a = b ≃ 155, c ≃ 138 Å, and diffracted X-rays to 2.5 Å resolution. The crystals were obtained using full-length Cel7A, which consists of a large 434-residue N-terminal catalytic domain capable of cleaving cellulose, a 27-residue flexible linker and a small 36-residue C-terminal carbohydrate-binding module (CBM). However, a preliminary analysis of the electron-density maps suggests that the linker and CBM are disordered in both crystal forms. Complete refinement and structure analysis are currently in progress.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2014; 70(Pt 6):773-776. · 0.55 Impact Factor
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    ABSTRACT: Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. D-Xylose isomerase carries out the first reaction in the catabolism of D-xylose, but is also able to convert D-glucose to D-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed. Here, the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data, is reported. Detailed analysis of the hydrogen-bond networks at the active site of the enzyme suggests that His54, which is doubly protonated, is poised to protonate the glucose O5 position, while Lys289, which is neutral, promotes deprotonation of the glucose O1H hydroxyl group via an activated water molecule. The structure also reveals an extended hydrogen-bonding network that connects the conserved residues Lys289 and Lys183 through three structurally conserved water molecules and residue 186, which is a glutamic acid to glutamine mutation.
    Acta Crystallographica Section D Biological Crystallography 02/2014; 70(Pt 2):414-420. · 12.67 Impact Factor
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    ABSTRACT: Members of the periplasmic binding protein (PBP) superfamily utilize a highly conserved inter-domain ligand binding site that adapts to specifically bind a chemically diverse range of ligands. This paradigm of PBP ligand binding specificity was recently altered when the structure of the Thermotoga maritima cellobiose-binding protein (tmCBP) was solved. The tmCBP binding site is bipartite, comprising a canonical solvent-excluded region (subsite one), adjacent to a solvent-filled cavity (subsite two) where specific and semi-specific ligand recognition occur, respectively. A molecular level understanding of binding pocket adaptation mechanisms that simultaneously allow both ligand specificity at subsite one and promiscuity at subsite two has potentially important implications in ligand binding and drug design studies. We sought to investigate the determinants of ligand binding selectivity in tmCBP through biophysical characterization of tmCBP in the presence of varying beta-glucan oligosaccharides. Crystal structures show that whilst the amino acids that comprise both the tmCBP subsite one and subsite two binding sites remain fixed in conformation regardless of which ligands are present, the rich hydrogen bonding potential of water molecules may facilitate the ordering and the plasticity of this unique PBP binding site. The identification of the roles these water molecules play in ligand recognition suggests potential mechanisms that can be utilized to adapt a single ligand binding site to recognize multiple distinct ligands.
    BMC Structural Biology 10/2013; 13(1):18. · 2.10 Impact Factor
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    ABSTRACT: The first high-resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory is reported. Neutron diffraction data extending to 1.65 Å resolution were collected from a relatively small 0.7 mm(3) PfRd crystal using 2.5 d (60 h) of beam time. The refined structure contains 371 out of 391, or 95%, of the D atoms of the protein and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolution (1.5 Å) from crystals with volume <1.0 mm(3) and with unit-cell edges <100 Å. Beamline features include novel elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm focal spot at the sample position with full-width vertical and horizontal divergences of 0.5 and 0.6°, respectively. Variable short- and long-wavelength cutoff optics provide automated exchange between multiple-wavelength configurations (λmin = 2.0, 2.8, 3.3 Å to λmax = 3.0, 4.0, 4.5, ∼20 Å). These optics produce a more than 20-fold increase in the flux density at the sample and should help to enable more routine collection of high-resolution data from submillimetre-cubed crystals. Notably, the crystal used to collect these PfRd data was 5-10 times smaller than those previously reported.
    Acta Crystallographica Section D Biological Crystallography 10/2013; 69(Pt 10):2157-2160. · 12.67 Impact Factor
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    ABSTRACT: Neutron scattering is exquisitely sensitive to the position, concentration, and dynamics of hydrogen atoms in materials and is a powerful tool for the characterization of structure-function and interfacial relationships in biological systems. Modern neutron scattering facilities offer access to a sophisticated, nondestructive suite of instruments for biophysical characterization that provides spatial and dynamic information spanning from Ångstroms to microns and from picoseconds to microseconds, respectively. Applications in structural biology range from the atomic-resolution analysis of individual hydrogen atoms in enzymes through to meso- and macro-scale analysis of complex biological structures, membranes, and assemblies. The large difference in neutron scattering length between hydrogen and deuterium allows contrast variation experiments to be performed and enables H/D isotopic labeling to be used for selective and systematic analysis of the local structure, dynamics, and interactions of multi-component systems. This overview describes the available techniques and summarizes their practical application to the study of biomolecular systems. Curr. Protoc. Protein Sci. 72:17.16.1-17.16.34. © 2013 by John Wiley & Sons, Inc.
    Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 04/2013; Chapter 17:Unit17.16.
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    ABSTRACT: Techniques and equipment have been developed that enable large protein crystals (1–6 mm 3) flash-cooled in liquid nitrogen at 77 K to be transferred and mounted on a liquid helium Displex cryorefrigerator and cooled to temperatures down to 15 K for accurate neutron diffraction analysis. In preliminary experiments, it was possible to collect high-quality high-resolution neutron diffraction data to 1.55 Å resolution from several large crystals of triclinic hen egg white lysozyme cooled to 15 K. This enabled the subsequent cryogenic analysis of two further proteins, rubredoxin and concanavalin A, at 1.7 and 2.5 Å , respectively, demonstrating the generality of the approach. The ability to flash-cool such large crystals for cryogenic neutron analysis should significantly broaden the range of scientific questions examined by neutron protein crystallography, allowing the analysis of structures and transitions as a function of temperature and enabling freeze-trapped capture of kinetic intermediates in protein systems.
    Journal of Applied Crystallography 08/2012; 45(4):686-692. · 3.34 Impact Factor
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    ABSTRACT: Neutron crystallography is a powerful technique for experimental visualization of the positions of light atoms, including hydrogen and its isotope deuterium. In recent years, structural biologists have shown increasing interest in the technique as it uniquely complements X-ray crystallographic data by revealing the positions of D atoms in macromolecules. With this regained interest, access to macromolecular neutron crystallography beamlines is becoming a limiting step. In this report, it is shown that a rapid data-collection strategy can be a valuable alternative to longer data-collection times in appropriate cases. Comparison of perdeuterated rubredoxin structures refined against neutron data sets collected over hours and up to 5 d shows that rapid neutron data collection in just 14 h is sufficient to provide the positions of 269 D atoms without ambiguity.
    Acta Crystallographica Section D Biological Crystallography 01/2012; 68(Pt 1):35-41. · 12.67 Impact Factor
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    ABSTRACT: A high-flux, 40m long small-angle neutron scattering (SANS) instrument has been constructed at Oak Ridge National Laboratory (ORNL). The facility utilizes a mechanical velocity selector, pinhole collimation and a high count-rate (> 105 Hz), large-area (1m2) two-dimensional position-sensitive detector. The incident wavelength ( ), resolution ( / ), incident collimation and sample-detector distance are independently variable under computer control. The detector can translate 45cm off axis to increase the overall Q-range (< 0.001 < Q = 4 -1sin < 1 -1), where 2 is the angle of scatter. The design and characteristics of this instrument are described along with examples of scattering data to illustrate the performance.
    Journal of Applied Crystallography 01/2012; 45(5). · 3.34 Impact Factor
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    ABSTRACT: Proteins are dynamic objects, constantly undergoing conformational fluctuations, yet the linkage between internal protein motion and function is widely debated. This study reports on the characterization of temperature-activated collective and individual atomic motions of oxidized rubredoxin, a small 53 residue protein from thermophilic Pyrococcus furiosus (RdPf). Computational modeling allows detailed investigations of protein motions as a function of temperature, and neutron scattering experiments are used to compare to computational results. Just above the dynamical transition temperature which marks the onset of significant anharmonic motions of the protein, the computational simulations show both a significant reorientation of the average electrostatic force experienced by the coordinated Fe(3+) ion and a dramatic rise in its strength. At higher temperatures, additional anharmonic modes become activated and dominate the electrostatic fluctuations experienced by the ion. At 360 K, close to the optimal growth temperature of P. furiosus, simulations show that three anharmonic modes including motions of two conserved residues located at the protein active site (Ile7 and Ile40) give rise to the majority of the electrostatic fluctuations experienced by the Fe(3+) ion. The motions of these residues undergo displacements which may facilitate solvent access to the ion.
    The Journal of Physical Chemistry B 05/2011; 115(28):8925-36. · 3.61 Impact Factor
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    ABSTRACT: AbstractSingle-crystal neutron diffraction data were collected at 20K to a resolution of 1.05Å on a crystal of the inverse formyl peptide receptor agonist cyclosporin H, CsH, (crystal form II, CsH-II) on the Laue diffractometer VIVALDI at the Institut Laue-Langevin (Grenoble). The solvent structure and hydrogen bonding network of CsH-II have been unambiguously determined by single-crystal neutron diffraction; the agreement factor R(F 2) is 13.5% for all 2726 reflections. All hydrogen atom positions, including methyl-group orientations, have been determined by crystallographic refinement. The neutron structure of cyclosporin provides unique and complementary insights into methyl orientation, hydrogen-bonding, and solvent interactions that are not available from X-ray analysis alone. Index AbstractCsH neutron structure showing the 7 waters and trace of the main chain N1---N11. All water hydrogens were determined experimentally. Thermal ellipsoids are plotted at 85% probability. Drawn with ORTEP-III/RASTER (Burnett and Johnson, Report ORNL-6895, 1996; Merritt and Bacon, Methods in Enzymology 277:505, 1997) as implemented in the program suite WinGX (Farrugia, Journal of Applied Crystallography 32(4):837, 1999) and generated by ORTEP-3 for Windows (Farrugia, Journal of Applied Crystallography 30(5):565, 1997). The main chain trace was drawn with RASMOL (Sayle, Glaxo research and development, 1994). KeywordsCyclosporin H–Neutron structure–Water hydrogens–Hydrogen bonding–Laue diffraction
    Journal of Chemical Crystallography 01/2011; 41(4):470-480. · 0.51 Impact Factor
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    ABSTRACT: In this report the single-particle scattering properties of five Platonic solids, including tetrahedron, hexahedron, octahedron, dodecahedron and icosahedron, are investigated in a systematic manner. For each given geometry, the Debye spatial autocorrelation function (r), pair distance distribution function (PDDF) p (r) and intraparticle structure factor (form factor) P (Q) are respectively calculated and compared to the corresponding scattering function of the spherical referential system. Based on our theoretical models, the empirical relationship between the dodecahedral and icosahedral structural characteristics and those of the equivalent spheres is found. Moreover, the single-particle scattering properties of the icosahedral and the spherical shells with the same volume are further investigated and the prospect of using different data analysis approaches to explore their structural difference is also presented and discussed.
    Journal of Applied Crystallography 01/2011; 44(3). · 3.34 Impact Factor
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    ABSTRACT: Lignocellulosic biomass, which is an abundant renewable natural resource, has the potential to play a major role in the generation of renewable biofuels through its conversion to bioethanol. Unfortunately, it is a complex biological composite material that shows significant recalcitrance, making it a cost-ineffective feedstock for bioethanol production. Small-angle neutron scattering (SANS) was employed to probe the multi-scale structure of cellulosic materials. Cellulose was extracted from milled native switchgrass and from switchgrass that had undergone a dilute acid pretreatment method in order to disrupt the lignocellulose structure. The high-Q structural feature (Q > 0.07 Å(-1)) can be assigned to cellulose fibrils based on a comparison of cellulose purified by solvent extraction of native and dilute acid pretreated switchgrass and a commercial preparation of microcrystalline cellulose. Dilute acid pretreatment results in an increase in the smallest structural size, a decrease in the interconnectivity of the fibrils and no change in the smooth domain boundaries at length scales larger than 1000 Å.
    Acta Crystallographica Section D Biological Crystallography 11/2010; 66(Pt 11):1189-93. · 12.67 Impact Factor
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    ABSTRACT: The generation of bioethanol from lignocellulosic biomass holds great promise for renewable and clean energy production. A better understanding of the complex mechanisms of lignocellulose breakdown during various pretreatment methods is needed to realize this potential in a cost and energy efficient way. Here we use small-angle neutron scattering (SANS) to characterize morphological changes in switchgrass lignocellulose across molecular to submicrometer length scales resulting from the industrially relevant dilute acid pretreatment method. Our results demonstrate that dilute acid pretreatment increases the cross-sectional radius of the crystalline cellulose fibril. This change is accompanied by removal of hemicellulose and the formation of R(g) ∼ 135 A lignin aggregates. The structural signature of smooth cell wall surfaces is observed at length scales larger than 1000 A, and it remains remarkably invariable during pretreatment. This study elucidates the interplay of the different biomolecular components in the breakdown process of switchgrass by dilute acid pretreatment. The results are important for the development of efficient strategies of biomass to biofuel conversion.
    Biomacromolecules 09/2010; 11(9):2329-35. · 5.37 Impact Factor
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    ABSTRACT: The locations of H atoms in biological structures can be difficult to determine using X-ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 A resolution neutron diffraction studies of fully perdeuterated and selectively CH(3)-protonated perdeuterated crystals of Pyrococcus furiosus rubredoxin (D-rubredoxin and HD-rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 A resolution X-ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the sigma level at the D-atom coordinate in simulated-annealing composite D-OMIT maps. It is shown that 1.65 A resolution RT neutron data for perdeuterated rubredoxin are approximately 8 times more likely overall to provide high-confidence positions for D atoms than 1.1 A resolution X-ray data at 100 K or RT. At or above the 1.0sigma level, the joint X-ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D-atom positions for D-rubredoxin and HD-rubredoxin, respectively. The X-ray-only 1.1 A resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D-atom positions above the 1.0sigma level for D-rubredoxin and HD-rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron-density maps, permitting the location of more D atoms than electron-density maps from models refined against X-ray data only.
    Acta Crystallographica Section D Biological Crystallography 05/2010; 66(Pt 5):558-67. · 12.67 Impact Factor
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    ABSTRACT: The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis virus has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis viruses grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a nondestructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling showed that, while the radial position of the lipid bilayer did not change significantly, it was possible to conclude that it did have significantly more cholesterol when the virus was grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis virus. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-cell-grown virus than in the virus from insect cells.
    Journal of Virology 03/2010; 84(10):5270-6. · 5.08 Impact Factor
  • Acta Crystallographica Section D Biological Crystallography 01/2010; D66(11):1189-1193. · 14.10 Impact Factor
  • Biomacromolecules 01/2010; 11(9):2329-2335. · 5.37 Impact Factor
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    ABSTRACT: A preliminary neutron crystallographic study of the proteolytic enzyme proteinase K is presented. Large hydrogenated crystals were prepared in deuterated crystallization buffer using the vapor-diffusion method. Data were collected to a resolution of 2.3 A on the LADI-III diffractometer at the Institut Laue-Langevin (ILL) in 2.5 d. The results demonstrate the feasibility of a full neutron crystallographic analysis of this structure with the aim of providing relevant information on the location of H atoms, particularly at the active site. This information will contribute to further understanding of the molecular mechanisms underlying the catalytic activity of proteinase K and to an enriched understanding of the subtilisin clan of serine proteases.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 03/2009; 65(Pt 2):184-7. · 0.55 Impact Factor

Publication Stats

764 Citations
329.91 Total Impact Points


  • 2005–2014
    • Oak Ridge National Laboratory
      • • Biology and Soft Matter Division
      • • Neutron Sciences Directorate
      Oak Ridge, Florida, United States
  • 2003–2011
    • Institut Laue-Langevin
      Grenoble, Rhône-Alpes, France
  • 2009–2010
    • North Carolina State University
      • Department of Molecular and Structural Biochemistry
      Raleigh, North Carolina, United States
  • 2006
    • University at Buffalo, The State University of New York
      Buffalo, New York, United States
  • 2000–2004
    • University of Southampton
      • Biological Sciences
      Southampton, ENG, United Kingdom
  • 1990–1996
    • Keele University
      • School of Life Sciences
      Newcastle under Lyme, ENG, United Kingdom