Dean A. A. Myles

Three dimensional structure of human C-reactive protein.

The structure of the classical acute phase reactant human C-reactive protein provides evidence that phosphocholine binding is mediated through calcium and a hydrophobic pocket centred on Phe 66. The residue Glu 81 is suitably positioned to interact with the choline group. A cleft on the pentameric face opposite to that containing the calcium site may have an important functional role. The structure provides insights into the molecular mechanisms by which this highly conserved plasma protein, for which no polymorphism or deficiency state is known, may exert its biological role.

Breakdown of Cell Wall Nanostructure in Dilute Acid Pretreated Biomass

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.

VIVALDI—A thermal-neutron laue diffractometer for physics, chemistry and materials science

Abstract VIVALDI is a Laue diffractometer based on a cylindrical image-plate detector that will accept a variety of standard and adapted sample environments for fast single-crystal experiments in physics, chemistry and materials science. By using the single-crystal Laue technique with a large solid-angle detector and a thermal neutron beam, the two-dimensional projection of a large volume of reciprocal space of small-unit-cell materials can be seen in a single exposure. Complete structural data can thus be obtained in a time shorter by one to two orders of magnitude than for a monochromatic experiment, with only a modest loss in precision. The dramatically shortened data-acquisition time allows structural and magnetic phase transitions—which often result in complex incommensurable structure—to be observed and followed in detail as a function of temperature or pressure. VIVALDI is scheduled to be in routine operation by the end of 2001.

Structural characterization of crystals of α-glycine during anomalous electrical behaviour

The crystal structure of α-glycine has been investigated in the temperature range 288–427 K using neutron diffraction. The molecular structure does not change significantly and the putative crystallographic phase transition associated with anomalous electrical behaviour in this temperature range is not observed. The unit cell expands anisotropically with increasing temperature, with the unique monoclinic b axis, corresponding to the stacking direction of molecular layers, changing the most. The increasing separation of antiferroelectric molecular layers with increasing temperature is driven by an increase in molecular libration about an axis that lies perpendicular to the b axis. There is also a weakening of the interlayer hydrogen bonds with temperature. These structural and dynamic changes will affect the response of molecular dipoles to an applied electric field and provide a possible mechanism for the anomalous electrical behaviour.

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 Å, 100K; 0.80 Å, 15K; 1.75 Å, 293K), neutron Laue data (2.2 Å, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.

Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050.

Integral membrane proteins are solubilized by their incorporation into a detergent micelle. The detergent micelle has a critical influence on the formation of a three-dimensional crystal lattice. The bulk detergent phase is not seen in X-ray crystal structures of integral membrane proteins, due to its disordered character. Here, we describe the detergent structure present in crystals of the peripheral light-harvesting complex of the purple bacteria Rhodopseudomonas acidophila strain 10050 at a maximal resolution of 12A as determined by neutron crystallography. The LH2 molecule has a toroidal shape and spans the membrane completely in vivo. A volume of 16% of the unit cell could be ascribed to detergent tails, localized on both the inner and outer hydrophobic surfaces of the molecule. The detergent tail volumes were found to be associated with individual LH2 molecules and had no direct role in the formation of the crystalline lattice.

High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

Mechanical Properties of Nanoscopic Lipid Domains

The lipid raft hypothesis presents insights into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. As a result, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approaches with inelastic neutron scattering, we isolate the bending modulus of ∼13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. From additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes.

The Bio-SANS instrument at the High Flux Isotope Reactor of Oak Ridge National Laboratory

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 instruments user community engages in.

The IMAGINE instrument: first neutron protein structure and new capabilities for neutron macromolecular crystallography.

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.