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. STATEMENT OF SIGNIFICANCE: The tropocollagen spring mechanism allows collagen fibrils from some tissues to elongate significantly under small loads, and its recent discovery has the potential to change our fundamental understanding of how tissue deforms. This time-resolved study quantifies the contribution of the spring mechanism to the local strain in stretched tissue and compares it to the contribution associated with the straightening of fibril waviness, the widely accepted primary low-load strain mechanism. The spring mechanism contributed more to the local tissue strain than fibril straightening, and was found to be elastic while fibril straightening was more viscous. The results suggest that the viscoelastic behaviour of a biomaterial is controlled, at least in part, by the relative amount of fibril-scale crimp and tropocollagen supramolecular twist.

VMXi: a fully automated, fully remote, high-flux in situ macromolecular crystallography beamline.

VMXi is a new high-flux microfocus macromolecular crystallography beamline at Diamond Light Source. The beamline, dedicated to fully automated and fully remote data collection of macromolecular crystals in situ, allows rapid screening of hundreds of crystallization plates from multiple user groups. Its main purpose is to give fast feedback at the complex stages of crystallization and crystal optimization, but it also enables data collection of small and delicate samples that are particularly difficult to harvest using conventional cryo-methods, crystals grown in the lipidic cubic phase, and allows for multi-crystal data collections in drug discovery programs. The beamline is equipped with two monochromators: one with a narrow band-pass and fine energy resolution (optimal for regular oscillation experiments), and one with a wide band-pass and a high photon flux (optimal for fast screening). The beamline has a state-of-the-art detector and custom goniometry that allows fast data collection. This paper describes the beamline design, current status and future plans.

Dynamics of P-type ATPase transport revealed by single-molecule FRET.

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.

Optimisation of recombinant production of active human cardiac SERCA2a ATPase.

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca(2+) translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.

An x-ray scattering study into the structural basis of corneal refractive function in an avian model.

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3-4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.

Crystal structure of the sodium-potassium pump.

The Na+,K+-ATPase generates electrochemical gradients for sodium and potassium that are vital to animal cells, exchanging three sodium ions for two potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 A resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the alpha-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The beta- and gamma-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices alphaM7/alphaM10 and alphaM9, respectively. The gamma-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the alpha-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.

Ca2+ versus Mg2+ coordination at the nucleotide-binding site of the sarcoplasmic reticulum Ca2+-ATPase.

The recently determined crystal structure of the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) with a bound ATP analogue (AMPPCP) reveals a compact state, similar to that found in the presence of ADP and aluminium fluoride. However, although the two Ca2+-binding sites in the membrane are known to be occluded in the latter state, in the AMPPCP-bound state the Ca2+-binding sites are not occluded under conditions with physiological levels of Mg2+ and Ca2+. It has been shown that the high concentration (10 mM) of Ca2+ used for crystallization (in the presence of Mg2+) may be responsible for the discrepancy. To determine whether Ca2+ competes with Mg2+ and affects the nucleotide-binding site, we have subjected the AMPPCP and ADP:AlF4- bound forms to crystallographic analysis by anomalous difference Fourier maps, and we have compared AMPPCP-bound forms crystallized in the absence or in the presence of Mg2+. We found that Ca2+ rather than Mg2+ binds together with AMPPCP at the phosphorylation site, whereas the ADP:AlF4- complex is associated with two magnesium ions. These results address the structure of the phosphorylation site before and during phosphoryl transfer. The bound CaAMPPCP nucleotide may correspond to the activated pre-complex, formed immediately before phosphorylation, whereas the Mg(2)ADP:AlF4- transition state complex reflects the preference for Mg2+ in catalysis. In addition, we have identified a phosphatidylcholine lipid molecule bound at the cytosol-membrane interface.

New light for science: synchrotron radiation in structural medicine.

Macromolecular crystallography (MX) is a powerful method for obtaining detailed three-dimensional structural information about macromolecules. MX using synchrotron X-rays has contributed, significantly, to both fundamental and applied research, including the structure-based design of drugs to combat important diseases. New third-generation synchrotrons offer substantial improvements in terms of quality and brightness of the X-ray beams they produce. Important classes of macromolecules, such as membrane proteins (including many receptors) and macromolecular complexes, are difficult to obtain in quantity and to crystallise, which has hampered analysis by MX. Intensely bright X-rays from the latest synchrotrons will enable the use of extremely small crystals, and should usher in a period of rapid progress in resolving these previously refractory structures.

Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump.

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) belongs to the group of P-type ATPases, which actively transport inorganic cations across membranes at the expense of ATP hydrolysis. Three-dimensional structures of several transport intermediates of SERCA1a, stabilized by structural analogues of ATP and phosphoryl groups, are now available at atomic resolution. This has enabled the transport cycle of the protein to be described, including the coupling of Ca(2+) occlusion and phosphorylation by ATP, and of proton counter-transport and dephosphorylation. From these structures, Ca(2+)-ATPase gradually emerges as a molecular mechanical device in which some of the transmembrane segments perform Ca(2+) transport by piston-like movements and by the transmission of reciprocating movements that affect the chemical reactivity of the cytosolic globular domains.