Influence of Divalent Metal Ions on the Precipitation of the Plasma Protein Fibrinogen.

Fibrinogen nanofibers are very attractive biomaterials to mimic the native blood clot architecture. Previously, we reported the self-assembly of fibrinogen nanofibers in the presence of monovalent salts and have now studied how divalent salts influence fibrinogen precipitation. Although the secondary fibrinogen structure was significantly altered with divalent metal ions, morphological analysis revealed exclusively smooth fibrinogen precipitates. In situ monitoring of the surface roughness facilitated predicting the tendency of various salts to form fibrinogen fibers or smooth films. Analysis of the chemical composition revealed that divalent salts were removed from smooth fibrinogen films upon rinsing while monovalent Na+ species were still present in fibrinogen fibers. Therefore, we assume that the decisive factor controlling the morphology of fibrinogen precipitates is direct ion-protein contact, which requires disruption of the ion-surrounding hydration shells. We conclude that in fibrinogen aggregates, this mechanism is effective only for monovalent ions, whereas divalent ions are limited to indirect fibrinogen adsorption.

Phosphorylation of Fibronectin Influences the Structural Stability of the Predicted Interchain Domain.

As a key player in cell adhesion, the glycoprotein fibronectin is involved in the complex mechanobiology of the extracellular matrix. Although the function of many modules in the fibronectin molecule has already been understood, the structure and biological relevance of the C-terminal cross-linked region (CTXL) still remains unclear. It is known that fibronectin is only phosphorylated in the CTXL domain, but no results have been presented to date, which indicate a biological function based on this phosphorylation. For the first time, we introduce a structural model of the CTXL region in fibronectin, which we obtained by exhaustive replica exchange molecular dynamics simulations (TIGER2hs). The sampling revealed a conformational landscape of the dimerization module, and the global minimum state showed an umbrella-like module body and conspicuous structural region with two feet. We observed that the CTXL foot region exhibits a structural stability in its physiological state, which disappears upon changes in the phosphorylation state. Thus, our in silico studies enabled us to show that the flexibility of the CTXL region is guided by phosphorylation. These results indicate an in vivo function of the CTXL domain in protein binding and cell adhesion, which is controlled by phosphorylation.

Lysine residues control the conformational dynamics of beta 2-glycoprotein I.

One of the major problems in the study of the dynamics of proteins is the visualization of changing conformations that are important for processes ranging from enzyme catalysis to signaling. A protein exhibiting conformational dynamics is the soluble blood protein beta 2-glycoprotein I (beta2GPI), which exists in two conformations: the closed (circular) form and the open (linear) form. It is hypothesized that an increased proportion of the open conformation leads to the autoimmune disease antiphospholipid syndrome (APS). A characteristic feature of beta2GPI is the high content of lysine residues. However, the potential role of lysine in the conformational dynamics of beta2GPI has been poorly investigated. Here, we report on a strategy to permanently open up the closed protein conformation by chemical acetylation of lysine residues using acetic acid N-hydroxysuccinimide ester (NHS-Ac). Specific and complete acetylation was demonstrated by the quantification of primary amino groups with fluoraldehyde o-phthalaldehyde (OPA) reagent, as well as western blot analysis with an anti-acetylated lysine antibody. Our results demonstrate that acetylated beta2GPI preserves its secondary and tertiary structures, as shown by circular dichroism spectroscopy. We found that after lysine acetylation, the majority of proteins are in the open conformation as revealed by atomic force microscopy high-resolution images. Using this strategy, we proved that the electrostatic interaction of lysine residues plays a major role in stabilizing the beta2GPI closed conformation, as confirmed by lysine charge distribution calculations. We foresee that our approach will be applied to other lysine-rich proteins (e.g. histones) undergoing conformational transitions. For instance, conformational dynamics can be triggered by environmental conditions (e.g. pH, ion concentration, post-translational modifications, and binding of ligands). Therefore, our study may be relevant for investigating the equilibrium of protein conformations causing diseases.

Simulated and experimental force spectroscopy of lysozyme on silica.

The force spectra of proteins detaching from oxide surfaces measured by atomic force microscopy (AFM) often present complex patterns of peaks, which are difficult to correlate with individual bond-breaking events at the atomic scale. In this work we rationalize experimental AFM force spectra of hen-egg-white lysozyme detaching from silica by means of all-atom steered molecular dynamics (SMD) simulations. In particular, we demonstrate that the native tertiary structure of lysozyme is preserved if, and only if, its four intramolecular disulfide bridges are intact. Otherwise, the protein pulled off the surface undergoes severe unfolding, which is well captured by SMD simulations in explicit solvent. Implicit solvent simulations, on the contrary, wrongly predict protein unfolding even in the presence of S-S bridges, due to the lack of additional structural stabilization provided by the water's hydrogen-bond network within and surrounding the protein. On the basis of our combined experimental and theoretical findings, we infer that the rugged force spectra characteristic of lysozyme/silica interfaces are not due to the successive breaking of internal disulfide bonds leading to partial unfolding events. Rather, they reflect the detachment of several molecules bound to the same AFM tip, each anchored to the surface via multiple hydrogen and ionic bonds.

The role of water co-adsorption on the modification of ZnO nanowires using acetic acid.

Density functional theory (DFT) and Car-Parinello molecular dynamic simulations were employed to investigate the interaction of acetic acid with non-polar facets of ultra-thin ZnO nanowires. We consider both a dry and a water environment as well as different molecule coverages for the hydrated system. Our calculations reveal that the fully-covered nanowire is energetically favored in the aqueous environment at room temperature. We also identified a minor influence of liquid water on the denticity of the ligands for the fully modified system. However, a monodentate adsorption is expected for a half-covered nanowire due to strong ligand-water interactions.

Molecular dynamics simulations of the amino acid-ZnO (10-10) interface: a comparison between density functional theory and density functional tight binding results.

We investigate the adsorption behavior of four different amino acids (glutamine, glutamate, serine, cysteine) on the zinc oxide (101̄0) surface, comparing the geometry and energy associated with a number of different adsorption configurations. In doing this, we highlight the benefits and limits of using density-functional tight-binding (DFTB) with respect to standard density functional theory (DFT). The DFTB method is found to reliably reproduce the DFT adsorption geometries. Analysis of the adsorption configurations emphasizes the fundamental role of the first hydration layer in mediating the interactions between the amino acids and the surface. Direct surface-molecule bonds are found to form predominantly via the carboxylate groups of the studied amino acids. No surface-mediated chemical reactions are observed, with the notable exception of a proton transfer from the thiol group of cysteine to a hydroxyl group of the surface hydration layer. The adsorption energies are found to be dominated both by the formation of direct or indirect surface-molecule hydrogen bonds, but also by the rearrangement of the hydrogen-bond network in surface proximity in a non-intuitive way. Energetic comparisons between DFTB and DFT are made difficult on one side by the long time necessary to achieve convergence of potential energy values in MD simulations and on the other side by the necessity of including higher-order corrections to DFTB to obtain a good description of the hydrogen bond energetics. Overall, our results suggest that DFTB is a good reference method to set the correct chemical states and the initial geometries of hybrid biomolecule/ZnO systems to be simulated with non-reactive force fields.

Principles of Fibrinogen Fiber Assembly In Vitro.

Fibrinogen nanofibers hold great potential for applications in wound healing and personalized regenerative medicine due to their ability to mimic the native blood clot architecture. Although versatile strategies exist to induce fibrillogenesis of fibrinogen in vitro, little is known about the underlying mechanisms and the associated length scales. Therefore, in this manuscript the current state of research on fibrinogen fibrillogenesis in vitro is reviewed. For the first time, the manifold factors leading to the assembly of fibrinogen molecules into fibers are categorized considering three main groups: substrate interactions, denaturing and non-denaturing buffer conditions. Based on the meta-analysis in the review it is concluded that the assembly of fibrinogen is driven by several mechanisms across different length scales. In these processes, certain buffer conditions, in particular the presence of salts, play a predominant role during fibrinogen self-assembly compared to the surface chemistry of the substrate material. Yet, to tailor fibrous fibrinogen scaffolds with defined structure-function-relationships for future tissue engineering applications, it still needs to be understood which particular role each of these factors plays during fiber assembly. Therefore, the future combination of experimental and simulation studies is proposed to understand the intermolecular interactions of fibrinogen, which induce the assembly of soluble fibrinogen into solid fibers.

Simulation of adhesion forces and energies of peptides on titanium dioxide surfaces.

Force field molecular dynamics simulations on a decapeptide in contact with a rutile (100) surface in aqueous solution are reported. The peptide sequence is part of α(1)-collagen. Force-distance curves yield discrete peaks to the rupture of charged lysine and glutamate side chains from the surface according to the model of contact points. The rupture forces are 0.2-2.2 nN, and the values strongly depend on the charges of surface hydroxyl groups. Adhesion energies are evaluated from the areas of the rupture peaks. For proton charges of 0.4 and 1, adhesion energies between 40 and 190 kJ/mol were found being comparable to recent ab initio molecular dynamics results. Flips in the torsional angles of the peptide are observed during restrained desorption. The partial charges of hydroxyproline are revised, and the polarization of the C(ß)-C(γ) bond as well as the ring pucker conformations are taken into account. It is shown that transition from collagen to helix fold is more likely for hydroxyproline than for proline and might be relevant for differences in the properties of POG (proline, hydroxyproline, glycine) and PPG collagen sequences.

Atomistic Details of Chymotrypsin Conformational Changes upon Adsorption on Silica.

Adsorption of enzymes on solid surfaces may lead to conformational changes that reduce their catalytic conversion activity and are thus detrimental to the efficiency of biotechnology or biosensing applications. This work is a joint theoretical and experimental endeavor in which we identify and quantify the conformational changes that chymotrypsin undergoes when in contact with the surface of amorphous silica nanoparticles. For this purpose, we use circular dichroism spectroscopy, standard molecular dynamics, and advanced-sampling methods. Only the combination of these techniques allowed us to pinpoint a destabilization effect of silica on specific structural motifs of chymotrypsin. They are linked by the possibility of theoretically predicting CD spectra, allowing us to elucidate the source of the experimentally observed spectral changes. We find that chymotrypsin loses part of its helical content upon adsorption, with minor perturbation of its overall tertiary structure, associated with changes in the aromatic interactions. We demonstrate that the C-terminal helical fragment is unfolded as an isolated oligopeptide in pure water, folded as an α-helix as terminus of chymotrypsin in solution, and again partly disordered when the protein is adsorbed on silica. We believe that the joint methodology introduced in this manuscript has a direct general applicability to investigate any biomolecule-inorganic surface system. Methods to theoretically predict circular dichroism spectra from atomistic simulations were compared and improved. The drawbacks of the approaches are discussed; in particular, the limited capability of advanced-sampling MD schemes to explore the conformational phase space of large proteins and the dependency of the predicted ellipticity bands on the choice of calculation parameters.

Physisorption of α-chymotrypsin on SiO2 and TiO2: A comparative study via experiments and molecular dynamics simulations.

In order to understand fundamental interactions at the interface between immobilized enzymes and ceramic supports, the authors compare the adsorption features of chymotrypsin on SiO2 and TiO2 colloidal particles by means of a combination of adsorption experiments and molecular dynamics simulations. While the dependency of the adsorption amount on pH is consistent with the trend predicted the Derjaguin-Landau-Verwey-Overbeek theory, other effects can only be rationalized if the atomic-scale details of the water-mediated protein-surface interactions are considered. On both surfaces, a clear driving force for the formation of a double monolayer at the saturation coverage is found. Although nearly equal free energies of adsorption are estimated on the two materials via a Langmuir adsorption analysis, about 50% more proteins per unit of surface can be accommodated on TiO2 than on SiO2. This is probably due to the lower surface diffusion mobility of the adsorbed protein in the latter case. Surface anchoring is realized by a combination of direct ionic interactions between charged proteins and surface sites (more pronounced for SiO2) and distinct structuring of the surface hydration layers in which the contact residues are embedded (more pronounced for TiO2). Finally, normalization of the data with respect to particle surface areas accessible to the proteins, rather than determined by means of the Brunauer-Emmett-Teller nitrogen adsorption isotherm, is crucial for a correct interpretation of the results.

Label-free biosensing with single-molecule force spectroscopy.

We demonstrate here a novel single-molecule, label-free bioanalytical system capable of sensing the presence of specific ssDNA oligomer sequences and proteins with high selectivity and sensitivity. An ssDNA concentration of 1 nM and a Lyz concentration of 0.65 nM could be detected.

Atomistic Simulations of the ZnO(12Ì 10)/Water Interface: A Comparison between First-Principles, Tight-Binding, and Empirical Methods.

We investigate the adsorption behavior of water over the zinc oxide (12̅10) surface starting from single molecules up to bulk liquid by means of atomistic molecular dynamics simulations. We compare results obtained with density-functional theory, density-functional tight binding, and a recently developed reactive force field. The methods perform comparably up to the level of a single monolayer of adsorbed water, predicting only small differences in adsorption energies and, as a consequence, adsorption geometries. These lie within the error bars of typical quantum mechanical calculations performed with different exchange-correlation functionals. However, the discrepancies among the methods have a dramatic effect on the dissociation equilibria and the structuring of liquid water layers in contact with the surface. Especially the different treatment of electrostatic interactions via self-consistent atomic point charges appears to heavily influence the simulation outcomes. Critical comparisons with experimental studies and possibly ad hoc reparametrizations of the semiempirical functionals may thus be necessary to study phenomena such as dissolution or biomolecular adsorption at ZnO surfaces within statistically relevant time and size scales.