Biomedical subjects

Clair Baldock

Publications and source records attributed to Clair Baldock.

16 recordsLinked to original sources

PubMed2006

The morphology of adsorbed extracellular matrix assemblies is critically dependent on solution calcium concentration.

The adsorption of proteins to surfaces may alter their biological properties. Understanding and controlling these interactions is important in ultrastructural, biochemical and cellular studies. We have previously demonstrated that both the morphology and biological function of extracellular matrix assemblies such as fibrillin and type VI collagen microfibrils are influenced by surface chemistry. In this study we have employed atomic force microscopy to determine if the morphology of extracellular matrix microfibrils is influenced by solution chemistry. Microfibrils were adsorbed to mica or poly-L-lysine modified mica (mica-PLL) in the presence of 31 microM-1000 microM Ca(2+). Although both microfibrillar species adsorbed to mica and mica-PLL at all calcium concentrations, maximal adsorption was observed on mica at 125-250 microM. On mica surfaces fibrillin microfibril morphology varied continuously with calcium concentration from laterally diffuse assemblies at high concentrations to compact assemblies at low concentrations. In contrast, distinct type VI collagen microfibril morphologies were observed at high, intermediate and low calcium concentrations. Similar calcium dependent microfibrillar morphologies were evident on mica-PLL. Therefore physiologically relevant concentrations of solution calcium, independent of surface charge, profoundly influenced both the adsorbed amount and morphology of native extracellular assemblies. These studies highlight the importance not only of surface chemistry but also of solute composition and concentration in influencing the morphology and hence biological function of adsorbed proteins.

Adsorption↗

PubMed2006

Marfan syndrome-causing mutations in fibrillin-1 result in gross morphological alterations and highlight the structural importance of the second hybrid domain.

Mutations in fibrillin-1 result in Marfan syndrome, which affects the cardiovascular, skeletal and ocular systems. The multiorgan involvement and wide spectrum of associated phenotypes highlights the complex pathogenesis underlying Marfan syndrome. To elucidate the genotype to phenotype correlations, we engineered four Marfan syndrome causing mutations into a fibrillin-1 fragment encoded by exons 18-25, a region known to interact with tropoelastin. Biophysical and biochemical approaches, including small angle x-ray scattering, analytical ultracentrifugation, and circular dichroism, were used to study the impact of these mutations upon the structure and function of the protein. Mutations G880S, C862R, and C908R, situated within the second hybrid domain, disrupted the ratio of alpha-helix to beta-sheet leading to a more compact conformation. These data clearly demonstrate the importance of the previously uncharacterized hybrid domain in fibrillin-1 structure. In contrast, mutation K1023N situated within the linker region between the third eight cysteine motif and cbEGF 11 markedly extended the length of the fragment. However, none of the mutations affected tropoelastin binding. The profound effects of all four mutations on fragment conformation suggest that they contribute to the pathogenesis of Marfan syndrome by disrupting protein folding and its assembly into fibrillin-rich microfibrils.

Amino Acid Sequence↗

PubMed2006

Nanostructure of fibrillin-1 reveals compact conformation of EGF arrays and mechanism for extensibility.

Fibrillin-1 is a 330-kDa multidomain extracellular matrix protein that polymerizes to form 57-nm periodic microfibrils, which are essential for all tissue elasticity. Fibrillin-1 is a member of the calcium-binding EGF repeat family and has served as a prototype for structural analyses. Nevertheless, both the detailed structure of fibrillin-1 and its organization within microfibrils are poorly understood because of the complexity of the molecule and the resistance of EGF arrays to crystallization. Here, we have used small-angle x-ray scattering and light scattering to analyze the solution structure of human fibrillin-1 and to produce ab initio structures of overlapping fragments covering 90% of the molecule. Rather than exhibiting a uniform rod shape as current models predict, the scattering data revealed a nonlinear conformation of calcium-binding EGF arrays in solution. This finding has major implications for the structures of the many other EGF-containing extracellular matrix and membrane proteins. The scattering data also highlighted a very compact, globular region of the fibrillin-1 molecule, which contains the integrin and heparan sulfate-binding sites. This finding was confirmed by calculating a 3D reconstruction of this region using electron microscopy and single-particle image analysis. Together, these data have enabled the generation of an improved model for microfibril organization and a previously undescribed mechanism for microfibril extensibility.

Epidermal Growth Factor↗

PubMed2006

Structural correlation between collagen VI microfibrils and collagen VI banded aggregates.

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.

Aged↗

PubMed2006

Tissue specific differences in fibrillin microfibrils analysed using single particle image analysis.

Fibrillin microfibrils endow mammalian connective tissues with elasticity and play a fundamental role in the deposition of elastin. The microfibrils are 57 nm periodic supramolecular protein polymers with a mass of 2.5 MDa per repeat. The organisation of molecules within a microfibril is still open to debate and structural studies are only just starting to unravel this issue. The contribution of microfibril associated proteins to microfibril ultrastructure and whether there are any tissue specific differences in microfibril structure is still unknown. Therefore, we have used low dose electron microscopy, single particle image analysis and atomic force microscopy to study the structure of fibrillin microfibrils from different tissues. EM images of microfibrils from aorta, ciliary zonules and vitreous humor were collected and more than 500 microfibril repeats from each sample were subjected to averaging. Averages from each sample were analysed using axial stain exclusion patterns and difference images to detect any variations between them. The overall morphology of fibrillin microfibrils was conserved between tissues and there were only very minor differences in the bead and shoulder region of microfibrils. These data suggest that the structure of isolated microfibrils represents the fibrillin scaffold, and either microfibril associated molecules are lost on purification or play only a minor role in microfibril structure.

Animals↗

PubMed2006

Reprint of "Structural correlation between collagen VI microfibrils and collagen VI banded aggregates" [J. Struct. Biol. 154 (2006) 312-326].

Journal Article↗

PubMed2005

Fibrillin-1 interactions with heparin. Implications for microfibril and elastic fiber assembly.

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four fibrillin-1 sites. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.

Animals↗

PubMed2005

Evidence for the intramolecular pleating model of fibrillin microfibril organisation from single particle image analysis.

Fibrillin microfibrils endow mammalian connective tissues with elasticity and are fundamental for the deposition of elastin. The microfibrils are 57nm periodic supramolecular protein polymers with a mass of 2.4MDa per repeat. The detailed structure and organisation of most matrix assemblies is poorly understood due to their large size and complexity and it has proved a major challenge to define their structural organisation. Therefore, we have used low dose electron microscopy and single particle image analysis to study the structure of fibrillin microfibrils. Three novel features were detected: a globular feature that bridges the "arm" region, a double band of density crossing the microfibril and stain penetrating holes present in the interbead region, possibly produced by the removal of microfibril associated proteins in the purification procedure. Fine filaments of approximately 2.4nm diameter are resolved in the interbead region, which correspond to the reported diameter of the fibrillin molecule. Comparison of the stain exclusion pattern of microfibrils with the theoretical stain exclusion pattern of fibrillin packing models indicates that the intramolecular pleating model, where each fibrillin molecule is pleated within one microfibril period allowing extensibility by unpleating, has the best fit to the data.

Animals↗

PubMed2005

Fibrillin microfibrils.

Fibrillin microfibrils are widely distributed extracellular matrix assemblies that endow elastic and nonelastic connective tissues with long-range elasticity. They direct tropoelastin deposition during elastic fibrillogenesis and form an outer mantle for mature elastic fibers. Microfibril arrays are also abundant in dynamic tissues that do not express elastin, such as the ciliary zonules of the eye. Mutations in fibrillin-1-the principal structural component of microfibrils-cause Marfan syndrome, a heritable disease with severe aortic, ocular, and skeletal defects. Isolated fibrillin-rich microfibrils have a complex 56 nm "beads-on-a-string" appearance; the molecular basis of their assembly and elastic properties, and their role in higher-order elastic fiber formation, remain incompletely understood.

Aging↗

PubMed2004

Homotypic fibrillin-1 interactions in microfibril assembly.

We have defined the homotypic interactions of fibrillin-1 to obtain new insights into microfibril assembly. Dose-dependent saturable high affinity binding was demonstrated between N-terminal fragments, between furin processed C-terminal fragments, and between these N- and C-terminal fragments. The N terminus also interacted with a downstream fragment. A post-furin cleavage site C-terminal sequence also interacted with the N terminus, with itself and with the furin-processed fragment. No other homotypic fibrillin-1 interactions were detected. Some terminal homotypic interactions were inhibited by other terminal sequences, and were strongly calcium-dependent. Treatment of an N-terminal fragment with N-ethylmaleimide reduced homotypic binding. Microfibril-associated glycoprotein-1 inhibited N- to C-terminal interactions but not homotypic N-terminal interactions. These fibrillin-1 interactions are likely to regulate pericellular fibrillin-1 microfibril assembly.

Amino Acid Motifs↗

PubMed2003

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.

Aged↗

PubMed2003

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.

Animals↗

PubMed2003

The supramolecular organization of collagen VI microfibrils.

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.

Animals↗

PubMed2002

Fibrillin: from microfibril assembly to biomechanical function.

Fibrillins form the structural framework of a unique and essential class of extracellular microfibrils that endow dynamic connective tissues with long-range elasticity. Their biological importance is emphasized by the linkage of fibrillin mutations to Marfan syndrome and related connective tissue disorders, which are associated with severe cardiovascular, ocular and skeletal defects. These microfibrils have a complex ultrastructure and it has proved a major challenge both to define their structural organization and to relate it to their biological function. However, new approaches have at last begun to reveal important insights into their molecular assembly, structural organization and biomechanical properties. This paper describes the current understanding of the molecular assembly of fibrillin molecules, the alignment of fibrillin molecules within microfibrils and the unique elastomeric properties of microfibrils.

Animals↗

PubMed2002

Crystallization and preliminary X-ray crystallographic studies on acyl-(acyl carrier protein) from Escherichia coli.

Acyl carrier proteins carry the lipid substrate to the enzymes of the fatty acid synthase system. Crystals of Escherichia coli acyl carrier protein to which a butyryl group has been attached via a thioester link to the phosphopantetheine prosthetic arm have been obtained by the hanging-drop vapour-diffusion method. These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 27.6, b = 41.6, c = 63.7 A. The asymmetric unit appears to contain one subunit, corresponding to a packing density of 2.1 A(3) Da(-1). Crystals of the selenomethionine-substituted (SeMet) protein were obtained using different conditions and belong to space group P6(3), with unit-cell parameters a = b = 63.4, c = 37.0 A, alpha = beta = 90, gamma = 120 degrees and with a monomer in the asymmetric unit (V(M) = 2.5 A(3) Da(-1)). Crystals of a SeMet butyryl-ACP I62M variant were obtained using the conditions for the native protein. Like the native crystals, these belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 27.3, b = 41.9, c = 64.5 A. A data set suitable for MAD phasing was collected from the crystals of the I62M variant to 1.8 A resolution on the ESRF beamline ID14-4.

Acyl Carrier Protein↗

PubMed2002

X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site.

Acyl carrier protein (ACP) is an essential cofactor in biosynthesis of fatty acids and many other reactions that require acyl transfer steps. We have determined the first crystal structures of an acylated form of ACP from E. coli, that of butyryl-ACP. Our analysis of the molecular surface of ACP reveals a plastic hydrophobic cavity in the vicinity of the phosphopantethylated Ser36 residue that is expanded and occupied by the butyryl and beta-mercaptoethylamine moieties of the acylated 4'-phosphopantetheine group in one of our crystal forms. In the other form, the cavity is contracted, and we propose that the protein has adopted the conformation after delivery of substrate into the active site of a partner enzyme.

Acyl Carrier Protein↗