Insights into hydrogen bonding and stacking interactions in cellulose.

In this quantum chemical study, we explore hydrogen bonding (H-bonding) and stacking interactions in different crystalline cellulose allomorphs; namely, cellulose I(ß) and cellulose III(I). We consider a model system representing a cellulose crystalline core made from six cellobiose units arranged in three layers with two chains per layer. We calculate the contributions of intrasheet and intersheet interactions to the structure and stability in both cellulose I(ß) and cellulose III(I) crystalline cores. Reference structures for this study were generated from molecular dynamics simulations of water-solvated cellulose I(ß) and III(I) fibrils. A systematic analysis of various conformations describing different mutual orientations of cellobiose units is performed using the hybrid density functional theory with the M06-2X with 6-31+G(d,p) basis sets. We dissect the nature of the forces that stabilize the cellulose I(ß) and cellulose III(I) crystalline cores and quantify the relative strength of H-bonding and stacking interactions. Our calculations demonstrate that individual H-bonding interactions are stronger in cellulose I(ß) than in cellulose III(I); however, the total H-bonding contribution to stabilization is larger in cellulose III(I) because of the highly cooperative nature of the H-bonding network. In addition, we observe a significant contribution from cooperative stacking interactions to the stabilization of cellulose I(ß). The theory of atoms-in-molecules (AIM) has been employed to characterize and quantify these intermolecular interactions. AIM analyses highlight the role of nonconventional CH···O H-bonding in the cellulose assemblies. Finally, we calculate molecular electrostatic potential maps for the cellulose allomorphs that capture the differences in chemical reactivity of the systems considered in our study.

Preliminary joint neutron time-of-flight and X-ray crystallographic study of human ABO(H) blood group A glycosyltransferase.

The biosyntheses of oligosaccharides and glycoconjugates are conducted by glycosyltransferases. These extraordinarily diverse and widespread enzymes catalyze the formation of glycosidic bonds through the transfer of a monosaccharide from a donor molecule to an acceptor molecule, with the stereochemistry about the anomeric carbon being either inverted or retained. Human ABO(H) blood group A α-1,3-N-acetylgalactosaminyltransferase (GTA) generates the corresponding antigen by the transfer of N-acetylgalactosamine from UDP-GalNAc to the blood group H antigen. To understand better how specific active-site-residue protons and hydrogen-bonding patterns affect substrate recognition and catalysis, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection and time-of-flight neutron diffraction data were collected to 2.5 Šresolution at the PCS to ∼85% overall completeness, with complementary X-ray diffraction data collected from a crystal from the same drop and extending to 1.85 Šresolution. Here, the first successful neutron data collection from a glycosyltransferase is reported.

Pectus excavatum.

In pectus excavatum, a developmental chest-wall deformity that affects approximately one in 400 births, the sternum is depressed inward and the ribs protrude anteriorly. This deformity can compromise pulmonary and/or cardiac function. It can also produce a characteristic radiographic appearance on frontal radiographs, which may be mistaken for right-middle-lobe opacification from pneumonia or atelectasis. I present a case of pectus excavatum in a teenage female with characteristic imaging findings, and the disposition of the case.

Enzymes for carbon sequestration: neutron crystallographic studies of carbonic anhydrase.

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme that catalyzes the reversible hydration of CO(2) to form HCO(3)(-) and H(+) using a Zn-hydroxide mechanism. The first part of catalysis involves CO(2) hydration, while the second part deals with removing the excess proton that is formed during the first step. Proton transfer (PT) is thought to occur through a well ordered hydrogen-bonded network of waters that stretches from the metal center of CA to an internal proton shuttle, His64. These waters are oriented and ordered through a series of hydrogen-bonding interactions to hydrophilic residues that line the active site of CA. Neutron studies were conducted on wild-type human CA isoform II (HCA II) in order to better understand the nature and the orientation of the Zn-bound solvent (ZS), the charged state and conformation of His64, the hydrogen-bonding patterns and orientations of the water molecules that mediate PT and the ionization of hydrophilic residues in the active site that interact with the water network. Several interesting and unexpected features in the active site were observed which have implications for how PT proceeds in CA.

A joint x-ray and neutron study on amicyanin reveals the role of protein dynamics in electron transfer.

The joint x-ray/neutron diffraction model of the Type I copper protein, amicyanin from Paracoccus denitrificans was determined at 1.8 A resolution. The protein was crystallized using reagents prepared in D(2)O. About 86% of the amide hydrogen atoms are either partially or fully exchanged, which correlates well with the atomic depth of the amide nitrogen atom and the secondary structure type, but with notable exceptions. Each of the four residues that provide copper ligands is partially deuterated. The model reveals the dynamic nature of the protein, especially around the copper-binding site. A detailed analysis of the presence of deuterated water molecules near the exchange sites indicates that amide hydrogen exchange is primarily due to the flexibility of the protein. Analysis of the electron transfer path through the protein shows that residues in that region are highly dynamic, as judged by hydrogen/deuterium exchange. This could increase the rate of electron transfer by transiently shortening through-space jumps in pathways or by increasing the atomic packing density. Analysis of C-HX bonding reveals previously undefined roles of these relatively weak H bonds, which, when present in sufficient number can collectively influence the structure, redox, and electron transfer properties of amicyanin.

Preliminary time-of-flight neutron diffraction study of human deoxyhemoglobin.

Human hemoglobin (HbA) is an intricate system that has evolved to efficiently transport oxygen molecules (O(2)) from lung to tissue. Its quaternary structure can fluctuate between two conformations, T (tense or deoxy) and R (relaxed or oxy), which have low and high affinity for O(2), respectively. The binding of O(2) to the heme sites of HbA is regulated by protons and by inorganic anions. In order to investigate the role of the protonation states of protein residues in O(2) binding, large crystals of deoxy HbA (approximately 20 mm(3)) were grown in D(2)O under anaerobic conditions for neutron diffraction studies. A time-of-flight neutron data set was collected to 1.8 A resolution on the Protein Crystallography Station (PCS) at the spallation source run by Los Alamos Neutron Science Center (LANSCE). The HbA tetramer (64.6 kDa; 574 residues excluding the four heme groups) occupies the largest asymmetric unit (space group P2(1)) from which a high-resolution neutron data set has been collected to date.

New sources and instrumentation for neutrons in biology.

Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.

Neutron and X-ray structural studies of short hydrogen bonds in photoactive yellow protein (PYP).

Photoactive yellow protein (PYP) from Halorhodospira halophila is a soluble 14 kDa blue-light photoreceptor. It absorbs light via its para-coumaric acid chromophore (pCA), which is covalently attached to Cys69 and is believed to be involved in the negative phototactic response of the organism to blue light. The complete structure (including H atoms) of PYP has been determined in D(2)O-soaked crystals through the application of joint X-ray (1.1 A) and neutron (2.5 A) structure refinement in combination with cross-validated maximum-likelihood simulated annealing. The resulting XN structure reveals that the phenolate O atom of pCA accepts deuterons from Glu46 O(epsilon2) and Tyr42 O(eta) in two unusually short hydrogen bonds. This arrangement is stabilized by the donation of a deuteron from Thr50 O(gamma1) to Tyr42 O(eta). However, the deuteron position between pCA and Tyr42 is only partially occupied. Thus, this atom may also interact with Thr50, possibly being disordered or fluctuating between the two bonds.

A preliminary time-of-flight neutron diffraction study on amicyanin from Paracoccus denitrificans.

Crystals of the blue copper protein amicyanin suitable for neutron diffraction were grown by the sitting-drop method, followed by repeated macroseeding using solutions prepared with D(2)O. Although the crystal sizes were the same, crystals grown using solutions made up in H(2)O in the initial stages of macroseeding and solutions with D(2)O in later stages did not diffract neutrons well. However, when the protein was initially exchanged with buffered D(2)O and then crystallized and also macroseeded using solutions made up in D(2)O throughout, the crystals diffracted neutrons to high resolution. One of those crystals was used to collect a data set to a resolution of 1.9 A.

X-ray structure of mercerized cellulose II at 1 a resolution.

A revised crystal structure for mercerized cellulose based on high-resolution synchrotron X-ray data collected from ramie fibers is reported (space group P2(1), a = 8.10(3) A, b = 9.03(3) A, c = 10.31(5) A, gamma = 117.10(5) degrees; 751 reflections in 304 composite spots; theta < 21.11 degrees; lambda = 0.7208 A; LALS refinement with d > 1.5 A, R' ' = 0.16; SHELX97 refinement with d > 1 A, R = 0.21). As with regenerated cellulose the crystal structure consists of antiparallel chains with different conformations but with the hydroxymethyl groups of both chains near the gt position. However, the conformation of the hydroxymethyl group of the center chain in the structure reported here differs significantly from the conformation in regenerated cellulose. This may be related to a large observed difference in the amount of hydroxymethyl group disorder: approximately 30% for regenerated cellulose and approximately 10% for mercerized cellulose.

A low-temperature neutron diffraction study of Mn12-acetate.

In the low-temperature region, where the dodecanuclear mixed-valence manganese carboxylate hexadecaacetatotetraaquadodecaoxododecamanganese bis(acetic acid) tetrahydrate, [Mn(12)O(12)(C(2)D(3)O(2))(16)(H(2)O)(4)].2C(2)HD(3)O(2).4H(2)O, displays unusual magnetic properties, its structure is similar to that previously determined at room temperature [Lis (1980). Acta Cryst. B36, 2042-2046], differing only by a small change in the configuration of one of the coordinated acetate groups, related to the formation of additional hydrogen bonds, and by the orientation of the methyl groups. Since most of the magnetization density of this system resides on the Mn atoms, the consequences of these rearrangements for the magnetic properties of the compound are small.

High resolution neutron fibre diffraction data on hydrogenated and deuterated cellulose.

High-resolution fibre neutron diffraction data were recorded from cellulose samples on a D19 diffractometer at the Institut Laue-Langevin (Grenoble). Highly crystalline cellulose I samples from Cladophora (cellulose I alpha + I beta) or Halocynthia (cellulose I beta) origin were prepared in the form of oriented films. Samples were studied in a hydrogenated form and in a hydrogen-deuterium exchanged deuterated form corresponding to all OH moieties being replaced by ODs. These samples, which diffracted to a resolution of around 0.9 A, gave diffraction diagrams consisting of several hundred independent diffraction spots. Crystalline cellulose II fibres resulting from the mercerization of flax were also studied in a hydrogenated form using NaOH/H2O as mercerizing medium and in a deuterated form using NaOD/D2O. Both of these samples diffracted to around 1.2 A, giving fibre diffraction diagrams slightly less resolved than those of cellulose I, but still consisting of more than one hundred independent diffraction spots. For cellulose I as well as for cellulose II, significant differences between the hydrogenated and deuterated patterns were observed and recorded. These new data should lead to improved structures for cellulose and direct identification of the position of hydrogen atoms involved in hydrogen bonding.

Neutron Laue diffraction studies of coenzyme cob(II)alamin.

Using a recently designed neutron single-crystal diffractometer utilizing a narrow-band Laue concept (LADI), diffraction data were collected from a crystal of the coenzyme cob(II)alamin (B12r), crystallized from a mixture of D2O and perdeuterated acetone. The instrument was placed at the end of a cold neutron guide at the Institute Laue Langevin (ILL, Grenoble, France), and data collection with neutrons of 1.8-8.0 A wavelength to a crystallographic resolution of 1.43 A was complete after about 36 h. This compares favourably with a previous experiment utilizing the same crystal specimen, where more than four weeks were required to collect monochromatic diffraction data to about 1 A resolution. Using the Laue data, the structure was solved by molecular replacement with the known X-ray crystal structure. Difference density maps revealed the atomic positions (including deuterium atoms) of seven ordered solvent water molecules and two (partially disordered) acetone molecules. These density maps were compared with corresponding maps computed with monochromatic neutron-diffraction data collected to 1. 0 A resolution using the same crystal specimen, as well as to maps derived from high-resolution (0.90 A) synchrotron X-ray data. In spite of the better definition of atomic positions in the two high-resolution maps, the 1.43 A LADI maps show considerable power for the determination of the location of hydrogen and deuterium positions.

Negative-pressure pulmonary edema: a complication of shoulder arthroscopy.

Negative-pressure pulmonary edema has been increasingly reported in surgical literature since 1977, yet there are no references to this condition in current orthopedic textbooks. We report a case of negative-pressure pulmonary edema occurring during arthroscopic surgery of the shoulder. The patient required reintubation for 5 days and hospitalization for 1 week. Because arthroscopic surgery is widely believed to be without complications, we want to make orthopedic surgeons aware of the possibility of negative-pressure pulmonary edema.

A high-angle neutron fibre diffraction study of the hydration of deuterated A-DNA.

A high-angle neutron fibre diffraction study of the hydration of A-DNA has been performed using the single-crystal diffractometer D19 at the Institut Laue-Langevin (Grenoble, France). The sample was prepared using deuterated DNA extracted from E. Coli cells cultured on deuterated nutrients. In common with our previous neutron fibre diffraction studies of DNA, this work exploits the ability to isotopically replace H2O around the DNA by D2O. However this study benefitted additionally from the fact that the hydrogen atoms which are covalently bonded to carbon atoms in the DNA sugars and bases were replaced by deuterium so that incoherent scattering and absorption effects were minimised. Successive cycles of Fourier synthesis and Fourier difference synthesis allowed water peaks to be identified and their positional and occupancy parameters to be refined against the observed diffraction data. The results confirm the main hydration features noted in our earlier studies with a clear network of water running along the inside edge of the major groove linking successive OI phosphate oxygen atoms. The central core of water running along the axis of the double helix is very much clearer in this work. Additionally this study shows chains of ordered water lying in the centre of the major groove.

DNA hydration studied by neutron fiber diffraction.

The development of neutron high angle fiber diffraction to investigate the location of water around the deoxyribonucleic acid (DNA) double-helix is described. The power of the technique is illustrated by its application to the D and A conformations of DNA using the single crystal diffractometer, D19, at the Institut Laue-Langevin. Grenoble and the time of flight diffractometer, SXD, at the Rutherford Appleton ISIS Spallation Neutron Source. These studies show the existence of bound water closely associated with the DNA. The patterns of hydration in these two DNA conformations are quite distinct and are compared to those observed in X-ray single crystal studies of two-stranded oligodeoxynucleotides. Information on the location of water around the DNA double-helix from the neutron fiber diffraction studies is combined with that on the location of alkali metal cations from complementary X-ray high angle fiber diffraction studies at the Daresbury Laboratory SRS using synchrotron radiation. These analyses emphasize the importance of viewing DNA, water and ions as a single system with specific interactions between the three components and provide a basis for understanding the effect of changes in the concentration of water and ions in inducing conformational transitions in the DNA double-helix.

Time-of-flight Laue fiber diffraction studies of perdeuterated DNA.

The diffractometer SXD at the Rutherford Appleton Laboratory ISIS pulsed neutron source has been used to record high resolution time-of-flight Laue fiber diffraction data from DNA. These experiments, which are the first of their kind, were undertaken using fibers of DNA in the A conformation and prepared using deuterated DNA in order to minimise incoherent background scattering. These studies complement previous experiments on instrument D19 at the Institut Laue Langevin using monochromatic neutrons. Sample preparation involved drawing large numbers of these deuterated DNA fibers and mounting them in a parallel array. The strategy of data collection is discussed in terms of camera design, sample environment and data collection. The methods used to correct the recorded time-of-flight data and map it into the final reciprocal space fiber diffraction dataset are also discussed. Difference Fourier maps showing the distribution of water around A-DNA calculated on the basis of these data are compared with results obtained using data recorded from hydrogenated A-DNA on D19. Since the methods used for sample preparation, data collection and data processing are fundamentally different for the monochromatic and Laue techniques, the results of these experiments also afford a valuable opportunity to independently test the data reduction and analysis techniques used in the two methods.

Comparison of home and laboratory based monitoring of NPT using the Rigiscan; a preliminary report.

Laboratory based and home based monitoring of nocturnal penile tumescence (NPT) with the Rigiscan device was compared in a consecutive series of 100 men monitored in the laboratory, and a further consecutive series of 100 men monitored at home. Recordings at home were significantly shorter and involved more interruptions than laboratory based records. With subgroups, matched for diagnosis or the reporting of waking erections as well as age, laboratory monitoring tended to produce more sustained erections. However, in spite of the matching of subgroups, this trend may have resulted from aetiological rather than methodological differences, and further comparison of the two methods in the same subjects is now warranted. In the meantime, home monitoring should be used with diagnostic caution.