The hierarchical response of human corneal collagen to load

Fibrillar collagen in the human cornea is integral to its function as a transparent lens of precise curvature, and its arrangement is now well-characterised in the literature. While there has been considerable effort to incorporate fibrillar architecture into mechanical models of the cornea, the mechanical response of corneal collagen to small applied loads is not well understood. In this study the fibrillar and molecular response to tensile load was quantified using small and wide angle X-ray scattering (SAXS/WAXS), and digital image correlation (DIC) photography was used to calculate the local strain field that gave rise to the hierarchical changes. A molecular scattering model was used to calculate the tropocollagen tilt relative to the fibril axis and changes associated with applied strain. Changes were measured in the D-period, molecular tilt and the orientation and spacing of the fibrillar and molecular networks. These measurements were summarised into hierarchical deformation mechanisms, which were found to contribute at varying strains. The change in molecular tilt is indicative of a sub-fibrillar “spring-like” deformation mechanism, which was found to account for most of the applied strain under physiological and near-physiological loads. This deformation mechanism may play an important functional role in tissues rich in fibrils of high helical tilt, such as skin and cartilage

Controlled dehydration of a ruthenium complex-DNA crystal induces reversible DNA kinking

Hydration-dependent DNA deformation has been known since Rosalind Franklin recognised that the relative humidity of the sample had to be maintained to observe a single conformation in DNA fibre diffraction. We now report for the first time the crystal structure, at the atomic level, of a dehydrated form of a DNA duplex and demonstrate the reversible interconversion to the hydrated form at room temperature. This system, containing d(TCGGCGCCGA) in the presence of Λ-[Ru(TAP)2(dppz)]2+ (TAP = 1,4,5,8-tetraazaphenanthrene, dppz = dipyridophenazine), undergoes a partial transition from an A/B hybrid to the A-DNA conformation, at 84-79% relative humidity. This is accompanied by an increase in kink at the central step from 22° to 51°, with a large movement of the terminal bases forming the intercalation site. This transition is reversible on rehydration. Seven datasets, collected from one crystal at room temperature, show the consequences of dehydration at near-atomic resolution. This result highlights that crystals, traditionally thought of as static systems, are still dynamic and therefore can be the subject of further experimentation

MxCuBE: a synchrotron beamline control environment customized for macromolecular crystallography experiments

MxCuBE is a beamline control environment optimized for the needs of macromolecular crystallography. This paper describes the design of the software and the features that MxCuBE currently provides

Crystallization of quinoprotein glucose dehydrogenase variants and homologues by microseeding.

The soluble quinoprotein glucose dehydrogenase oxidizes glucose, maltose and a variety of other monosaccharides and disaccharides to the corresponding lactones. An efficient microseeding protocol is reported to produce crystals of three variants that display reduced activity towards maltose. Similar cross-seeding protocols to grow crystals of homologues from Escherichia coli and Streptomyces coelicolor are described

Membrane protein structure determination - the next generation.

The field of Membrane Protein Structural Biology has grown significantly since its first landmark in 1985 with the first three-dimensional atomic resolution structure of a membrane protein. Nearly twenty-six years later, the crystal structure of the beta2 adrenergic receptor in complex with G protein has contributed to another landmark in the field leading to the 2012 Nobel Prize in Chemistry. At present, more than 350 unique membrane protein structures solved by X-ray crystallography (http://blanco.biomol.uci.edu/mpstruc/exp/list, Stephen White Lab at UC Irvine) are available in the Protein Data Bank. The advent of genomics and proteomics initiatives combined with high-throughput technologies, such as automation, miniaturization, integration and third-generation synchrotrons, has enhanced membrane protein structure determination rate. X-ray crystallography is still the only method capable of providing detailed information on how ligands, cofactors, and ions interact with proteins, and is therefore a powerful tool in biochemistry and drug discovery. Yet the growth of membrane protein crystals suitable for X-ray diffraction studies amazingly remains a fine art and a major bottleneck in the field. It is often necessary to apply as many innovative approaches as possible. In this review we draw attention to the latest methods and strategies for the production of suitable crystals for membrane protein structure determination. In addition we also highlight the impact that third-generation synchrotron radiation has made in the field, summarizing the latest strategies used at synchrotron beamlines for screening and data collection from such demanding crystals. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding

Structural basis for MOF and MSL3 recruitment into the dosage compensation complex by MSL1

The male-specific lethal (MSL) complex is required for dosage compensation in Drosophila melanogaster, and analogous complexes exist in mammals. We report structures of binary complexes of mammalian MSL3 and the histone acetyltransferase (HAT) MOF with consecutive segments of MSL1. MSL1 interacts with MSL3 as an extended chain forming an extensive hydrophobic interface, whereas the MSL1-MOF interface involves electrostatic interactions between the HAT domain and a long helix of MSL1. This structure provides insights into the catalytic mechanism of MOF and enables us to show analogous interactions of MOF with NSL1. In Drosophila, selective disruption of Msl1 interactions with Msl3 or Mof severely affects Msl1 targeting to the body of dosage-compensated genes and several high-affinity sites, without affecting promoter binding. We propose that Msl1 acts as a scaffold for MSL complex assembly to achieve specific targeting to the X chromosome

Tropocollagen springs allow collagen fibrils to stretch elastically

The mechanical properties of connective tissues are tailored to their specific function, and changes can lead to dysfunction and pathology. In most mammalian tissues the mechanical environment is governed by the micro- and nano-scale structure of collagen and its interaction with other tissue components, however these hierarchical properties remain poorly understood. In this study we use the human cornea as a model system to characterise and quantify the dominant deformation mechanisms of connective tissue in response to cyclic loads of physiological magnitude. Synchronised biomechanical testing, x-ray scattering and 3D digital image correlation revealed the presence of two dominant mechanisms: collagen fibril elongation due to a largely elastic, spring-like straightening of tropocollagen supramolecular twist, and a more viscous straightening of fibril crimp that gradually increased over successive loading cycles. The distinct mechanical properties of the two mechanisms suggest they have separate roles in vivo. The elastic, spring-like mechanism is fast-acting and likely responds to stresses associated with the cardiac cycle, while the more viscous crimp mechanism will respond to slower processes, such as postural stresses. It is anticipated that these findings will have broad applicability to understanding the normal and pathological functioning of other connective tissues such as skin and blood vessels that exhibit both helical structures and crimp

Effects of mineralization on the hierarchical organization of collagen—a synchrotron X-ray scattering and polarized second harmonic generation study

The process of mineralization fundamentally alters collagenous tissue biomechanics. While the structure and organization of mineral particles have been widely studied, the impact of mineralization on collagen matrix structure, particularly at the molecular scale, requires further investigation. In this study, synchrotron X-ray scattering (XRD) and polarization-resolved second harmonic generation microscopy (pSHG) were used to study normally mineralizing turkey leg tendon in tissue zones representing different stages of mineralization. XRD data demonstrated statistically significant differences in collagen D-period, intermolecular spacing, fibril and molecular dispersion and relative supramolecular twists between non-mineralizing, early mineralizing and late mineralizing zones. pSHG analysis of the same tendon zones showed the degree of collagen fibril organization was significantly greater in early and late mineralizing zones compared to non-mineralizing zones. The combination of XRD and pSHG data provide new insights into hierarchical collagen–mineral interactions, notably concerning possible cleavage of intra- or interfibrillar bonds, occlusion and reorganization of collagen by mineral with time. The complementary application of XRD and fast, label-free and non-destructive pSHG optical measurements presents a pathway for future investigations into the dynamics of molecular scale changes in collagen in the presence of increasing mineral deposition

Effects of mineralization on the hierarchical organization of collagen—a synchrotron X-ray scattering and polarized second harmonic generation study

The process of mineralization fundamentally alters collagenous tissue biomechanics. While the structure and organization of mineral particles have been widely studied, the impact of mineralization on collagen matrix structure, particularly at the molecular scale, requires further investigation. In this study, synchrotron X-ray scattering (XRD) and polarization-resolved second harmonic generation microscopy (pSHG) were used to study normally mineralizing turkey leg tendon in tissue zones representing different stages of mineralization. XRD data demonstrated statistically significant differences in collagen D-period, intermolecular spacing, fibril and molecular dispersion and relative supramolecular twists between non-mineralizing, early mineralizing and late mineralizing zones. pSHG analysis of the same tendon zones showed the degree of collagen fibril organization was significantly greater in early and late mineralizing zones compared to non-mineralizing zones. The combination of XRD and pSHG data provide new insights into hierarchical collagen–mineral interactions, notably concerning possible cleavage of intra- or interfibrillar bonds, occlusion and reorganization of collagen by mineral with time. The complementary application of XRD and fast, label-free and non-destructive pSHG optical measurements presents a pathway for future investigations into the dynamics of molecular scale changes in collagen in the presence of increasing mineral deposition