Elastin enables the reversible deformation of elastic tissues and can withstand decades of repetitive forces. Tropoelastin is the soluble precursor to elastin, the main elastic protein found in mammals. Little is known of the shape and mechanism of assembly of tropoelastin as its unique composition and propensity to self-associate has hampered structural studies. In this study, we solve the nanostructure of full-length and corresponding overlapping fragments of tropoelastin using small angle X-ray and neutron scattering, allowing us to identify discrete regions of the molecule. Tropoelastin is an asymmetric coil, with a protruding foot that encompasses the C-terminal cell interaction motif. We show that individual tropoelastin molecules are highly extensible yet elastic without hysteresis to perform as highly efficient molecular nanosprings. Our findings shed light on how biology uses this single protein to build durable elastic structures that allow for cell attachment to an appended foot. We present a unique model for head-to-tail assembly which allows for the propagation of the molecule's asymmetric coil through a stacked spring design.AFM | SAXS | atomic force microscopy A ll mammals rely on elastin to convey extensional elasticity to their tissues. Elastin dominates the mass of the aorta where it encounters the peaks and troughs of systole and diastole over the course of two billion heartbeats in a lifetime (1). The lung expands with each intake of breath and elastically contracts on exhalation. The function of these tissues benefits from minimal energy loss during elastic return in each cycle of expansion and contraction. Additionally, elastin is required to function in an environment that relies on cellular contact (2-4) without compromising persistent elasticity. This high level of physical performance demanded of elastin vastly exceeds and indeed outlasts all human-made elastomers (5).Elastin is constructed by the hierarchical assembly and crosslinking of many tropoelastin monomers that accumulate on a microfibrillar skeleton. Tropoelastin is encoded by a single gene in humans and predominantly laid down in utero and early childhood, providing a durable resource that is intended to elastically serve until old age. This exquisite assembly helps to generate elastic tissues as diverse as artery, lung, and skin (4). Consequences of elastolytic damage in aortic aneurysms, emphysema, and solar elastosis confirm the key roles of elastin in structure and cellular interactions (6-8). These tissues rely on this paradoxical combination of organized tissue structures built from an intrinsically unstructured protein. Tropoelastin serves as a component of rigidly organized assemblies, yet enables the formation of dynamically distensible, elastic tissues.Tropoelastin is frequently described in the literature as an unstructured protein, mainly because models of elasticity invoke an element of disorder within the structure (4, 9, 10). While this concept appears to be the case at the fine, more subtle intramolecular leve...

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.elastic fibers ͉ fibrillin microfibrils ͉ solution x-ray scattering

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 ␣-helix to ␤-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.

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Purpose: It is thought that proteoglycan (PG) alterations, collagen matrix reorganisation and the onset of corneal transparency in the developing avian cornea might be related events. The current histochemical study was designed to establish the character and distribution of corneal PG filaments in relation to collagen organisation during tissue morphogenesis. Methods: Corneas from days 13–18 developing chicken embryos were treated with cuprolinic blue (CuB) to examine sulphated PGs by transmission electron microscopy and quantitative image analysis. Results: On developmental day 13, corneas contained poorly defined lamellae and a large number of both small and large CuB-stained PG filaments, randomly distributed and often in collagen-free regions. By day 14 and after, the large CuB-stained PG filaments were much less abundant. At this time, too, collagen fibrils displayed an axial alignment and an occasional periodic arrangement of small CuB-stained PG filaments along their axes. By developmental day 15, lamellae were well formed and continued to increase in number and size thereafter. Between developmental days 16 and 17, there was a significant increase (p < 0.001) in the number of small, collagen-associated PG filaments. This increase persisted into day 18. Conclusions: The size, number and distribution of sulphated, CuB-stained PG filaments in the developing avian cornea change over time. This is particularly true between developmental days 13 and 14 and between days 16 and 17, concurrent with previously documented structural changes.

Techniques for global optimization (which include Monte Carlo and simulated annealing methods, neural networks, genetic algorithms, other evolutionary procedures, and a wide range of other computational strategies) are currently being applied to yield fundamental understanding across diverse areas of the physical sciences, [1] including protein folding, [ 2a] structure prediction of clusters [ 2b-e] and crystals, [ 2f] structure determination from electron diffraction [ 2g] and powder X-ray diffraction [3] data (including applications in the case of nanostructures [ 2h] ), interpretation of spectroscopic data such as NMR spectroscopy [ 2i] and Mçssbauer spectroscopy, [ 2j] understanding protein-substrate interactions, [ 2k] and optimization of laser pulse-shapes for quantum control of chemical reactions, [ 2l, m] to name just a few. Although the details of the computational implementations of global optimization strategies necessarily differs between these different areas of application, there is nonetheless significant commonality in the approaches employed in different fields, and fundamental advances in one field of application are often directly transferable to other fields.Among these areas of application, structure determination of organic molecular solids from powder X-ray diffraction data is nowadays carried out widely, [3] with the recent upsurge of activity in this field coinciding with the development of the "direct-space strategy" for structure solution [4] in which the structure-solution process is transformed into a problem of global optimization. Thus, in the direct-space strategy, trial structures are generated independently of the experimental powder X-ray diffraction pattern, and the quality of each trial structure is assessed by direct comparison between the powder X-ray diffraction pattern calculated for the trial structure and the experimental powder X-ray diffraction pattern. This comparison is quantified using an appropriate figure-ofmerit (in our work, the weighted powder profile R-factor R wp ). Clearly, the aim of the global optimization problem in this case is to locate the trial structure that corresponds to optimal agreement (lowest R wp ) between calculated and experimental powder X-ray diffraction patterns, and is equivalent to exploring a hypersurface R wp (G) to find the global minimum, where G represents the set of variables that define the trial structures. In principle, any technique for global optimization may be used, and our own current work in this field is focused on the use of a genetic algorithm, [5] implemented in the program EAGER.[6] Conventionally, the structural variables in the set G comprise, for each molecule in the asymmetric unit, the position {x, y, z} and orientation {q, f, y} of the whole molecule, and a set of n variable torsion angles {t 1 , t 2 ,.., t n } to define the molecular conformation. An important feature underlying the success of the direct-space strategy is that it incorporates reliable prior knowledge of molecular geometry (i.e....

The lateral spacing of fibril-forming collagen molecules does not change as the chick cornea develops between days 13 and 18. An orthogonal array of collagen fibrils is present in the corneas of developmental day-13 to -18 chicks, but starting at developmental day 16, additional collagen is deposited in a less well-oriented manner and thus acts to obscure the overall orthogonality, with implications for the biomechanical strength and shape of the cornea.

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