The purpose of this investigation was to determine what structural changes convert "inert" alphaIIbbeta3 integrins into "activated" high-affinity receptors for adhesive proteins. Light scattering, analytical ultracentrifugation, electron microscopy, and molecular modeling were used to probe the conformational states of the alphaIIbbeta3 integrin. Isolated from human blood platelets in octyl glucoside, the alphaIIbbeta3 complex behaved as an asymmetric 230 kDa macromolecule with a z-average translational diffusion coefficient of 2.9 F and a weight-average sedimentation coefficient of 7.7 S. Dynamic light scattering showed that ligand-mimetic peptides (RGDX, X = F, W, S) caused prompt, concentration-dependent increases in the Stokes radius (R(s)) of the alphaIIbbeta3 complex, whereas control peptides of reversed sequence (XDGR, X = F, W, S) had no significant effect. Sedimentation velocity data coupled with time-derivative analyses showed that RGDX peptides shifted the distribution of alphaIIbbeta3 sedimenting species toward smaller s values. Sedimentation equilibrium measurements indicated that a slower increase in the alphaIIbbeta3 molecular weight distribution took place in the presence of RGDX ligand-mimetics. Electron microscopy showed a split of alphaIIbbeta3's globular domain into two distinct nodules in the presence of RGDX peptides; oligomers joined through their stalk regions were seen frequently. These observations suggest that receptor occupancy by ligand-mimetic RGDX peptides is tightly coupled to relatively large changes in the structure of the alphaIIbbeta3 complex. alphaIIbbeta3 bead models were developed to describe quantitatively the ligand-induced transition from a "closed" to an "open" integrin conformation and the limited oligomerization that follows. This provides a new mechanistic framework for understanding integrin activation and the formation of signaling clusters on the surface of stimulated platelets.

The structure and kinetics of fibrin gels grown from fibrinogen solutions under quasiphysiological conditions, but in absence of Ca++, were investigated by means of elastic light scattering. By combining classical light scattering and low-angle elastic light scattering, an overall wave-vector range of about three decades was spanned, from q approximately 3 x 10(2) to q approximately 3 x 10(5) cm(-1). The scattered intensity distribution of the gels was measured in absolute units and fitted to a single function, which was able to reproduce accurately the data over the entire wave-vector range. From the fitting, it was possible to estimate the average diameter d of the fibrin fibers, the average crossover length xi of the gel, and establish the fractal nature of the gel structure, with a measure of its fractal dimension D(m). The measure of the intensity in absolute units also allowed the estimate of the density rho of the fibrin fibers and provided an independent measure of their size. The kinetics of formation of the gel was described in terms of a simple growth model: the scaffold of the network is formed very early in the course of the gelation process, at a "networking time," t(n), which is much smaller than the time required to form the final gel. At times t>t(n), the gel structure remains substantially unchanged and the successive growth consists only in a thickening of the gel fibers. Gels prepared under the same physical-chemical conditions, but at different fibrinogen concentrations, exhibited rather similar structures and kinetics, showing that the modalities of the gelation process are mainly governed by the solution conditions, and only secondarily by the fibrinogen concentration. For gels at fibrinogen concentration of approximately 0.24 mg/ml, the gel parameters were d approximately 130 nm, xi approximately 27 microm, D(m) approximately 1.3, and rho approximately 0.4 g/cm(3). Our d and rho values are in very good agreement with electron microscopy- and turbidity-derived literature data, respectively, while xi seems to be related to the mesh size of the initial scaffold formed at t(n), rather than to the mesh size of the final aged gel.

The interpretation of solution hydrodynamic data in terms of macromolecular structural parameters is not a straightforward task. Over the years, several approaches have been developed to cope with this problem, the most widely used being bead modeling in various flavors. We report here the implementation of the SOMO (SOlution MOdeller; Rai et al. in Structure 13:723-734, 2005) bead modeling suite within one of the most widely used analytical ultracentrifugation data analysis software packages, UltraScan (Demeler in Modern analytical ultracentrifugation: techniques and methods, Royal Society of Chemistry, UK, 2005). The US-SOMO version is now under complete graphical interface control, and has been freed from several constraints present in the original implementation. In the direct beads-per-atoms method, virtually any kind of residue as defined in the Protein Data Bank (e.g., proteins, nucleic acids, carbohydrates, prosthetic groups, detergents, etc.) can be now represented with beads whose number, size and position are all defined in usereditable tables. For large structures, a cubic grid method based on the original AtoB program (Byron in Biophys J 72:408-415, 1997) can be applied either directly on the atomic structure, or on a previously generated bead model. The hydrodynamic parameters are then computed in the rigid-body approximation. An extensive set of tests was conducted to further validate the method, and the results are presented here. Owing to its accuracy, speed, and versatility, US-SOMO should allow to fully take advantage of the potential of solution hydrodynamics as a complement to higher resolution techniques in biomacromolecular modeling.Correspondence to: Mattia Rocco, mattia.rocco@istge.it.

The platelet integrin ␣IIb␤3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of ␣IIb␤3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for ␣IIb␤3. Eptifibatide binding promptly and reversibly perturbed the conformation of the ␣IIb␤3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptorperturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted ␣IIb␤3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected ␣IIb␤3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to ␣IIb␤3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects ␣IIb␤3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.

The US-SOMO HPLC-SAXS (high-performance liquid chromatography coupled with small-angle X-ray scattering) module is an advanced tool for the comprehensive analysis of SEC-SAXS (size-exclusion chromatography coupled with SAXS) data. It includes baseline and band-broadening correction routines, and Gaussian decomposition of overlapping skewed peaks into pure components.

Many biological supramolecular structures are formed by polymerization of macromolecular monomers. Light scattering techniques can provide structural information from such systems, if suitable procedures are used to collect the data and then to extract the relevant parameters. We present an experimental set-up in which a commercial multiangle laser light scattering photometer is linked to a stopped-flow mixer, allowing, in principle, the time-resolved extrapolation of the weight-average molecular weight M(w) and of the z-average square radius of gyration (z) of the polymers from Zimm-like plots. However, if elongated structures are formed as the polymerization proceeds, curved plots rapidly arise, from which M(w) and (z) cannot be recovered by linear fitting. To verify the correctness of a polynomial fitting procedure, polydisperse collections of rod-like or worm-like particles of different lengths, generated at various stages during bifunctional polycondensations of rod-like macromolecular monomers, were considered. Then, the angular dependence of their time-averaged scattered intensity was calculated in the Rayleigh-Gans-Debye approximation, with random and systematic noise also added to the data. For relatively narrow size distributions, a third-degree polynomial fitting gave satisfactory results across a broad range of conversion degrees, yielding M(w) and (z) values within 2% and no greater than 10-20%, respectively, of the calculated values. When more broad size distributions were analyzed, the procedure still performed well for semiflexible polymers, but started to seriously underestimate both M(w) and (z) when rigid rod-like particles were analyzed, even at relatively low conversion degrees. The data were also analyzed in the framework of the Casassa approximation, from which the mass per unit length of the polymers can be derived. These procedures were applied to a set of data taken on the early stages of the thrombin-catalyzed polymerization of fibrinogen, a rod-like macromolecule approximately 50 nm long. The polymers, grown in the absence of Ca(2+) by rate-limiting amounts of thrombin, appeared to be characterized by a much broader size distribution than the one expected for a classical Flory bifunctional polycondensation, and they seem to behave as relatively flexible worm-like double-stranded chains. Evidence for the formation of fibrinogen-fibrin monomer complexes is also inferred from the time dependence of the mass/length ratio. However, our data are also compatible with the presence of limited amounts of single-stranded structures in the very early stages, either as a secondary, less populated pathway, or as transient intermediates to the classical double-stranded fibrils.

The concentration dependence of the structure of fibrin gels, formed following fibrinogen activation by thrombin at a constant molar ratio, was investigated by means of elastic light scattering techniques. The scattered intensity distributions were measured in absolute units over a wave-vector range q of about three decades ( approximately 3x10(2)-3x10(5) cm(-1)). A set of gel-characterizing parameters were recovered by accurately fitting the data with a single function recently developed by us [F. Ferri et al., Phys. Rev. E 63, 031401 (2001)], based on a simple structural model. Accordingly, the gels can be described as random networks of fibers of average diameter d and density rho, entangled together to form densely packed and spatially correlated blobs of mass fractal dimension D(m) and average size (or crossover length) xi. As previously done for d, we show here that the recovered xi is also a good approximation of a weight average, namely, d approximately sqrt[(w)] and xi approximately (w). By varying the fibrinogen concentration c(F) between 0.034-0.81 mg/ml, gels with 100> or =xi> or =10 microm, 100< or =d< or =200 nm, 1.2< or =D(m)< or =1.4, and constant rho approximately 0.4 mg/ml were obtained. The power-law c(F) dependencies that we found for both xi and d are consistent with the model, provided that the blobs are allowed to partially overlap by a factor eta likewise scaling with c(F) (2> or =eta> or =1). Recasting the whole dataset on a single master curve provided further evidence of the similarity between the structures of all the gels, and confirmed the self-consistency of the model.

The radius of gyration of human plasma fibronectin was determined by light scattering both under conditions in which the molecule is in an extended conformation (ionic strength 1.01 M, pH 8) and close to its native, more compact conformation (ionic strength 0.16 M, pH 8). These values were found to be 17.5 +/‐ 0.8 nm and 10.7 +/‐ 0.9 nm respectively, for a constant mol. wt of 533,000 +/‐ 8000, in excellent agreement with the value of 520,000 deduced from its known composition. A set of models, each made of two identical, end‐to‐end joined chains of 28 beads, was then constructed, and their calculated physico‐chemical parameters were compared with those available for the whole fibronectin molecule and for some of its proteolytic fragments in both conformations. Two possible models for the circulating form are presented here: in both, the fibronectin molecule is in a compact, tangled conformation, with the amino‐terminal end of one chain folded over to the carboxy end of itself or of the other chain either in a hairpin or in a circular fashion. With the exception of the carboxy‐terminal fibrin(ogen)‐binding domains, all the domains appear to be well exposed to the solvent, and thus free to interact with potential ligands.