Structure of cellulose microfibrils in primary cell walls from collenchyma.

In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production.

The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil.

Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection.

An evaluation of the reactivity of synthetic and natural apatites in the presence of aqueous metals.

Metal removal from contaminated effluents was examined following reaction with natural apatites of biological and geological origin or a synthetic hydroxylapatite (HAP). Mammalian meat and bone meal (MBM), a by-product from meat industry, was the biological apatite source. The effect of incineration on metal removal capacity of MBM and HAP was also examined. The reactivity of apatites for all tested metals (Pb, Cd, Cu and Zn) followed the general order: synthetic > biological > mineral. For all apatites tested, Pb was removed best and preferentially from multi-metal solutions. MBM and HAP (0.5 g solid) removed Pb completely from both highly concentrated single metal solutions (50 ml, 1000 mg/L Pb) and from multi-metal solutions (50 ml) with 100 mg/L each of Cd, Cu and Zn in addition to Pb. The incineration of MBM (725 degrees C and 850 degrees C) reduced significantly its capacity for removal of Zn (by 47%, from 56 mg/g to 9 mg/g) and Cd (by 38%, from 53 mg/g to 13 mg/g) in particular and to a lesser extent for Cu (by 14%, from 61 mg/g to 46 mg/g) while the removal of Pb was not affected (100 mg/g). The same pattern was observed for incinerated HAP. SEM and XRD analysis indicated that HAP reacted with the metals by precipitation of pure metal phosphates--Pb hydroxylapatite, Zn phosphate (hopeite), a Cd phosphate (identified only by ED-SEM) and Cu phosphate (libenthenite).

Analysis of collagen fibril diameter distribution in connective tissues using small-angle X-ray scattering.

Analysis of the diameters of collagen fibrils provides insight into the structure and physical processes occurring in the tissue. This paper describes a method for analyzing the frequency distribution of the diameters of collagen fibrils from small-angle X-ray scattering (SAXS) patterns. Frequency values of fibril diameters were input into a mathematical model of the form factor to calculate the equatorial intensity which best fits the experimentally derived data from SAXS patterns. A minimization algorithm utilizing simulated annealing (SA) was used in the fitting procedure. The SA algorithm allowed for random sampling of the frequency values, and was run iteratively to build up an optimized frequency distribution of fibril diameters. Results were obtained for collagen samples from sheep spine ligaments. The mean fibril diameter value obtained from this data-fitting method was 73 nm+/-20 nm (S.D.). From scanning transmission electron microscopy, the mean diameter was found to be 69 nm+/-14 nm (S.D.). The good agreement between the two methods demonstrates the reliability of the SAXS method for the tissue examined. The non-destructive nature of this technique, as well as its statistical robusticity and capacity for large sampling, means that this method is both quick and effective.

Fibrillin microfibrils are stiff reinforcing fibres in compliant tissues.

Fibrillin-rich microfibrils have endowed tissues with elasticity throughout multicellular evolution. We have used molecular combing techniques to determine Young's modulus for individual microfibrils and X-ray diffraction of zonular filaments of the eye to establish the linearity of microfibril periodic extension. Microfibril periodicity is not altered at physiological zonular tissue extensions and Young's modulus is between 78 MPa and 96 MPa, which is two orders of magnitude stiffer than elastin. We conclude that elasticity in microfibril-containing tissues arises primarily from reversible alterations in supra-microfibrillar arrangements rather than from intrinsic elastic properties of individual microfibrils which, instead, act as reinforcing fibres in fibrous composite tissues.

Rapid shape determination of tissue transglutaminase using high-throughput computing.

Small-angle X-ray scattering can be used to determine the molecular shape of macromolecules in solution which are otherwise refractory to conventional high-resolution studies. DAMMIN and GASBOR are applications that utilize ab initio methods to build models of proteins using simulated annealing; both DAMMIN and GASBOR have to be run numerous times on the same input data to generate the most likely protein shape. Condor is a specialized workload-management system for PC computation-intensive tasks. Using Condor, DAMMIN and GASBOR can be run a number of times simultaneously on the same input data, allowing the shape of proteins to be determined in a fraction of the time it would have taken to have run DAMMIN and GASBOR sequentially. The main advantage of this approach is that it allows quicker data processing; therefore, results are obtained promptly and without undue delay. Tissue transglutaminase is a multidomain enzyme that catalyses the formation of isopeptide bonds between polypeptide chains. This reaction requires the enzyme to undergo a series of conformational changes that are not well understood in order to allow the sequential interaction with the two substrate proteins and their subsequent release when cross-linked. Condor was applied to determine the solution shape of tissue transglutaminase in a rapid fashion. Eventually, the next step will be to move towards online analysis at synchrotron sources by developing a graphical user interface that will enable remote access, allowing users to submit jobs to Condor whilst at synchrotrons.

The Laminin 511/521-binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3.

The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the alpha5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

Molecular phylogeny of the extinct cave lion Panthera leo spelaea.

To reconstruct the phylogenetic position of the extinct cave lion (Panthera leo spelaea), we sequenced 1 kb of the mitochondrial cytochrome b gene from two Pleistocene cave lion DNA samples (47 and 32 ky B.P.). Phylogenetic analysis shows that the ancient sequences form a clade that is most closely related to the extant lions from Africa and Asia; at the same time, cave lions appear to be highly distinct from their living relatives. Our data show that these cave lion sequences represent lineages that were isolated from lions in Africa and Asia since their dispersal over Europe about 600 ky B.P., as they are not found among our sample of extant populations. The cave lion lineages presented here went extinct without mitochondrial descendants on other continents. The high sequence divergence in the cytochrome b gene between cave and modern lions is notable.

Raman microscopy and X-ray diffraction, a combined study of fibrillin-rich microfibrillar elasticity.

Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular matrix of a wide variety of connective tissues. Microfibrils are characterized as beaded filamentous structures with a variable axial periodicity (average 56 nm in the untensioned state); however, the basis of their elasticity remains unknown. This study used a combination of small angle x-ray scattering and Raman microscopy to investigate further the packing of microfibrils within the intact tissue and to determine the role of molecular reorganization in the elasticity of these microfibrils. The application of relatively small strains produced no overall change in either molecular or macromolecular microfibrillar structure. In contrast, the application of larger tissue extensions (up to 150%) resulted in a markedly different structure, as observed by both Raman microscopy and small angle x-ray scattering. These changes occurred at different levels of architecture and are interpreted as ranging from alterations in peptide bond conformation to domain rearrangement. This study demonstrates the importance of molecular elasticity in the mechanical properties of fibrillin-rich microfibrils in the intact tissue.