Mattia Rocco

Models of fibronectin.

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.

Developments in the US-SOMO bead modeling suite: New features in the direct residue-to-bead method, improved grid routines, and influence of accessible surface area screening

The US-SOMO suite provides a flexible interface for accurately computing solution parameters from 3D structures of biomacromolecules through bead-modeling approaches. We present an extended analysis of the influence of accessible surface area screening, overlap reduction routines, and approximations for non-coded residues and missing atoms on the computed parameters for models built by the residue-to-bead direct correspondence and the cubic grid methods. Importantly, by taking the theoretical hydration into account at the atomic level, the performance of the grid-type models becomes comparable or exceeds that of the corresponding hydrated residue-to-bead models.

Small- and wide-angle elastic light scattering study of fibrin structure

We show how small- and wide-angle elastic light scattering (q ∼ 0.03-30 μm -1 ) can be used to quantitatively characterize the structure of polymeric gels made of semi-flexible entangled fibers. We applied the technique to the study of fibrin gels grown from the polymerization of fibrinogen (FG) macromolecular monomers following activation by the enzyme thrombin (TH), at different concentrations and under different physical-chemical conditions of the gelling solution. Our findings show that the gel can be imagined as a random network of fibers of size d and density p, entangled together to form densely packed blobs of mass fractal dimension D m and average size ξ, which may overlap by a factor η and exhibit a long-range order. Provided that d ≥50-100 nm, all of the above parameters can be recovered by the use of a global fitting function developed by us on the basis on the proposed gel model. When the fibers are thinner (d < ∼50 nm), only the fiber mass/length ratio μ ∼ ρd 2 can be retrieved instead of d and p. Our data confirm and quantify the major changes in the gel structure that can be obtained by varying either the salt concentration of the solution and/or the molar ratio TH/FG. Gels formed in Tris-HCl 50 mM/NaCl 150 mM, pH 7.4 and TH/FG = 0.01 are characterized by relatively small, fairly branched (D in ∼ 1.4-2.0) fibers with a mass/length ratio independent of concentration. On reducing the TH/FG ratio, the fibers become increasingly thicker, with d ∼ 90 nm at TH/FG = 10 -5 . When the salt concentration is reduced to NaCl 100 mM (TH/FG = 0.01) the fibers are less branched (D m ∼ 1.2-1.4), but much thicker, with μ increasing by an order of magnitude. These effects are quantitatively analyzed and discussed.

Fibrinogen species as resolved by HPLC-SAXS data processing within the UltraScan Solution Modeler (US-SOMO) enhanced SAS module

The usefulness of a new high-performance liquid chromatography/small-angle X-ray scattering (HPLC-SAXS) data analysis module within the multi-resolution modeling suite US-SOMO is illustrated with size-exclusion small-angle X-ray scattering (SE-SAXS) data of a crude bovine serum albumin sample. The module is then applied to the SE-SAXS study of a human plasma fibrinogen high-molecular-weight fraction presenting severe aggregation problems and a split non-symmetrical main elution peak probably resulting from in-column degradation.

Binding of a fibrinogen mimetic stabilizes integrin αIIbβ3's open conformation

The platelet integrin αIIbβ3 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 αIIbβ3s fibrinogen binding site affected the receptors 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 αIIbβ3. Eptifibatide binding promptly and reversibly perturbed the conformation of the αIIbβ3 complex. Ligand‐specific decreases in its diffusion and sedimentation coefficient were observed at near‐stoichiometric eptifibatide concentrations, in contrast to the receptor‐perturbing effects of RGD ligands that we previously observed only at a 70‐fold molar excess. Eptifibatide promoted αIIbβ3 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 αIIbβ3 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 αIIbβ3s extracellular fibrinogen‐recognition site stabilizes the receptors 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 αIIbβ3s conformation, our findings provide new mechanistic insights into ligand‐linked integrin activation, clustering and signaling.

Polymerization of Rod-Like Macromolecular Monomers Studied by Stopped-Flow, Multiangle Light Scattering: Set-Up, Data Processing, and Application to Fibrin Formation

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.

US-SOMO HPLC-SAXS module: dealing with capillary fouling and extraction of pure component patterns from poorly resolved SEC-SAXS data

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.

Integrin αIIbβ3:ligand interactions are linked to binding-site remodeling

This study tested the hypothesis that high‐affinity binding of macromolecular ligands to the αIIbβ3 integrin is tightly coupled to binding‐site remodeling, an induced‐fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between αIIbβ3 and two model ligands—echistatin, a 6‐kDa recombinant protein with an RGD integrin‐targeting sequence, and fibrinogens γ‐module, a 30‐kDa recombinant protein with a KQAGDV integrin binding site—were measured by sedimentation velocity, fluorescence anisotropy, and a solid‐phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the αIIb/β3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming—i.e., ectodomain conformational changes and oligomerization induced by incubation at 35°C with the ligand‐mimetic peptide cHarGD—promotes complex formation with fibrinogens γ‐module. We also observed that the γ‐modules flexible carboxy terminus was not required for αIIbβ3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding‐site remodeling must first occur. Echistatins binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.

Hydrodynamic and mass spectrometry analysis of nearly-intact human fibrinogen, chicken fibrinogen, and of a substantially monodisperse human fibrinogen fragment X

The shape and solution properties of fibrinogen are affected by the location of the C-terminal portion of the Aalpha chains, which is presently still controversial. We have measured the hydrodynamic properties of a human fibrinogen fraction with these appendages mostly intact, of chicken fibrinogen, where they lack 11 characteristic 13-amino acids repeats, and of human fragment X, a plasmin early degradation product in which they have been removed. The human fibrinogen/fragment X samples were extensively characterized by SDS-PAGE/Western blotting and mass spectrometry, allowing their composition to be precisely determined. The solution properties of all samples were then investigated by analytical ultracentrifugation and size-exclusion HPLC coupled with multi-angle light scattering and differential pressure viscometry detectors. The measured parameters suggest that the extra repeats have little influence on the overall fibrinogen conformation, while a significant change is brought about by the removal of the C-terminal portion of the Aalpha chains beyond residue Aalpha200.

The application of atmospheric pressure matrix-assisted laser desorption/ionization to the analysis of long-term cryopreserved serum peptidome

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API-MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API-MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at -80°C for 18 months or at -20°C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API-MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.