RESUMEN
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.
Asunto(s)
Elasticidad , Especificidad de Órganos , Tropoelastina/química , Animales , Entropía , Humanos , Modelos Moleculares , Difracción de Neutrones , Conformación Proteica , Dispersión del Ángulo Pequeño , Soluciones , Vertebrados/metabolismo , Difracción de Rayos XRESUMEN
PURPOSE: The molecular chaperone αB-crystallin is found in high concentrations in the lens and is present in all major body tissues. Its structure and the mechanism by which it protects its target protein from aggregating and precipitating are not known. METHODS: Dynamic light scattering and X-ray solution scattering techniques were used to investigate structural features of the αB-crystallin oligomer when complexed with target proteins under mild stress conditions, i.e., reduction of α-lactalbumin at 37 °C and malate dehydrogenase when heated at 42 °C. In this investigation, the size, shape and particle distribution of the complexes were determined in real-time following the induction of stress. RESULTS: Overall, it is observed that the mass distribution, hydrodynamic radius, and spherical shape of the αB-crystallin oligomer do not alter significantly when it complexes with its target protein. CONCLUSIONS: The data are consistent with the target protein being located in the outer protein shell of the αB-crystallin oligomer where it is readily accessible for possible refolding via the action of other molecular chaperones.
Asunto(s)
Lactalbúmina/metabolismo , Luz , Malato Deshidrogenasa/metabolismo , Desplegamiento Proteico , Dispersión de Radiación , Cadena B de alfa-Cristalina/metabolismo , Animales , Bovinos , Humanos , Lactalbúmina/química , Malato Deshidrogenasa/química , Estructura Cuaternaria de Proteína , Sus scrofa , Temperatura , Rayos X , Cadena B de alfa-Cristalina/químicaRESUMEN
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.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Antígenos CD/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Moléculas de Adhesión Celular/metabolismo , Adhesinas Bacterianas/química , Antígenos CD/química , Proteínas de la Membrana Bacteriana Externa/química , Sitios de Unión , Moléculas de Adhesión Celular/química , Dicroismo Circular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Receptores de Superficie Celular , TermodinámicaRESUMEN
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.
Asunto(s)
Proteínas/química , Transglutaminasas/química , Simulación por Computador , Cristalografía por Rayos X , Modelos Moleculares , Conformación ProteicaRESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Laminina/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Sitios de Unión , Moléculas de Adhesión Celular/genética , Cristalografía por Rayos X , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Inmunoglobulinas/química , Sistema del Grupo Sanguíneo Lutheran , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/genética , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Homología Estructural de ProteínaRESUMEN
The avian eggshell is a highly ordered calcitic bioceramic composite, with both inorganic and organic constituents. The interactions between the inorganic and organic components within the structure are poorly understood but are likely to occur at the nanometre level. Thus structural variation at this level may impinge on the overall structural integrity and mechanical performance of the eggshell, and therefore analysis at this level is fundamental in fully understanding this ordered structure. In this study, structural changes in the mineral crystallites were investigated by microfocus small-angle X-ray scattering (microSAXS) using synchrotron radiation. Small-angle X-ray scattering (SAXS) can be used to investigate structures on the nanometre scale such as size, shape, arrangement and internal porosity. A microfocused X-ray beam, 1.5 microm vertically by 7 microm, was used to produce vertical linear scans of the eggshell section. SAXS patterns were taken from the eggshell membrane (inner surface of the eggshell) to the cuticle (outer surface of the eggshell). This allowed textural variations within the eggshell to be mapped. The scattering intensity profile was then used to derive the dimension of scattering objects that define the nanotexture. The nanotexture observed may result from the presence of the organic matrix, which is embedded as intracrystalline particles producing voids within the calcified framework of large (>1 microm) calcite crystals. Porod analysis revealed the average size of a scattering interface to be approximately 4.5 nm with small changes that had a depth-dependent variation. These were largest at the mammillary layer/membrane boundary. The palisade layer displayed a small upward trend in size of scattering object. Parallel scans showed that the textural variations observed within the palisade layer are significant and indicate local subtextures. In addition, many of the patterns exhibit diffuse scattering streaks that could result from reflectivity from the larger crystallite interfaces. Changes in the orientation of diffuse streaks were observed within the different layers, the membranes, mammillary layer, palisade layer, vertical crystal layer and cuticle, indicating certain preferred orientations of the crystallites within the layers. The nanotextural variations that are apparent could have implications at the macroscopic level of the resulting eggshell.