Self-association of a highly charged arginine-rich cell-penetrating peptide.

Small-angle X-ray scattering (SAXS) measurements reveal a striking difference in intermolecular interactions between two short highly charged peptides-deca-arginine (R10) and deca-lysine (K10). Comparison of SAXS curves at high and low salt concentration shows that R10 self-associates, while interactions between K10 chains are purely repulsive. The self-association of R10 is stronger at lower ionic strengths, indicating that the attraction between R10 molecules has an important electrostatic component. SAXS data are complemented by NMR measurements and potentials of mean force between the peptides, calculated by means of umbrella-sampling molecular dynamics (MD) simulations. All-atom MD simulations elucidate the origin of the R10-R10 attraction by providing structural information on the dimeric state. The last two C-terminal residues of R10 constitute an adhesive patch formed by stacking of the side chains of two arginine residues and by salt bridges formed between the like-charge ion pair and the C-terminal carboxyl groups. A statistical analysis of the Protein Data Bank reveals that this mode of interaction is a common feature in proteins.

Structural Characterization of Histatin 5-Spermidine Conjugates: A Combined Experimental and Theoretical Study.

Histatin 5 (Hst5) is a naturally occurring antimicrobial peptide that acts as the first line of defense against oral candidiasis. It has been shown that conjugation of the active Hst5 fragment, Hst54-15, and the polyamine spermidine (Spd) improves the candidacidal effect. Knowledge about the structure of these conjugates is, however, very limited. Thus, the aim of this study was to characterize the structural properties of the Hst54-15-Spd conjugates by performing atomistic molecular dynamics simulations in combination with small-angle X-ray scattering. It was shown that the Hst54-15-Spd conjugates adopt extended and slightly rigid random coil conformations without any secondary structure in aqueous solution. It is hypothesized that the increased fungal killing potential of Hst54-15-Spd, in comparison with the Spd-Hst54-15 conjugate, is due to the more extended conformations of the former, which cause the bonded Spd molecule to be more accessible for recognition by polyamine transporters in the cell.

Coarse-grained modeling of the intrinsically disordered protein Histatin 5 in solution: Monte Carlo simulations in combination with SAXS.

Monte Carlo simulations and coarse-grained modeling have been used to analyze Histatin 5, an unstructured short cationic salivary peptide known to have anticandidical properties. The calculated scattering functions have been compared with intensity curves and the distance distribution function P(r) obtained from small angle X-ray scattering (SAXS), at both high and low salt concentrations. The aim was to achieve a molecular understanding and a physico-chemical insight of the obtained SAXS results and to gain information of the conformational changes of Histatin 5 due to altering salt content, charge distribution, and net charge. From a modeling perspective, the accuracy of the electrostatic interactions are of special interest. The used coarse-grained model was based on the primitive model in which charged hard spheres differing in charge and in size represent the ionic particles, and the solvent only enters the model through its relative permittivity. The Hamiltonian of the model comprises three different contributions: (i) excluded volumes, (ii) electrostatic, and (iii) van der Waals interactions. Even though the model can be considered as gross omitting all atomistic details, a great correspondence is obtained with the experimental results. Proteins 2016; 84:777-791. © 2016 Wiley Periodicals, Inc.

Dynamical Oligomerisation of Histidine Rich Intrinsically Disordered ProteinS Is Regulated through Zinc-Histidine Interactions.

Intrinsically disordered proteins (IDPs) can form functional oligomers and in some cases, insoluble disease related aggregates. It is therefore vital to understand processes and mechanisms that control pathway distribution. Divalent cations including Zn2+ can initiate IDP oligomerisation through the interaction with histidine residues but the mechanisms of doing so are far from understood. Here we apply a multi-disciplinary approach using small angle X-ray scattering, nuclear magnetic resonance spectroscopy, calorimetry and computations to show that that saliva protein Histatin 5 forms highly dynamic oligomers in the presence of Zn2+. The process is critically dependent upon interaction between Zn2+ ions and distinct histidine rich binding motifs which allows for thermodynamic switching between states. We propose a molecular mechanism of oligomerisation, which may be generally applicable to other histidine rich IDPs. Finally, as Histatin 5 is an important saliva component, we suggest that Zn2+ induced oligomerisation may be crucial for maintaining saliva homeostasis.

Utilizing Coarse-Grained Modeling and Monte Carlo Simulations to Evaluate the Conformational Ensemble of Intrinsically Disordered Proteins and Regions.

In this study, we have used the coarse-grained model developed for the intrinsically disordered saliva protein (IDP) Histatin 5, on an experimental selection of monomeric IDPs, and we show that the model is generally applicable when electrostatic interactions dominate the intra-molecular interactions. Experimental and theoretically calculated small-angle X-ray scattering data are presented in the form of Kratky plots, and discussions are made with respect to polymer theory and the self-avoiding walk model. Furthermore, the impact of electrostatic interactions is shown and related to estimations of the conformational ensembles obtained from computer simulations and "Flexible-meccano." Special attention is given to the form factor and how it is affected by the salt concentration, as well as the approximation of using the form factor obtained under physiological conditions to obtain the structure factor.

Molecular Dynamics Simulations of Intrinsically Disordered Proteins: Force Field Evaluation and Comparison with Experiment.

An increasing number of studies using molecular dynamics (MD) simulations of unfolded and intrinsically disordered proteins (IDPs) suggest that current force fields sample conformations that are overly collapsed. Here, we study the applicability of several state-of-the-art MD force fields, of the AMBER and GROMOS variety, for the simulation of Histatin 5, a short (24 residues) cationic salivary IDP with antimicrobial and antifungal properties. The quality of the simulations is assessed in three complementary analyses: (i) protein shape and size comparison with recent experimental small-angle X-ray scattering data; (ii) secondary structure prediction; (iii) energy landscape exploration and conformational class analysis. Our results show that, indeed, standard force fields sample conformations that are too compact, being systematically unable to reproduce experimental evidence such as the scattering function, the shape of the protein as compared with the Kratky plot, and intrapeptide distances obtained through the pair distance distribution function, p(r). The consistency of this deviation suggests that the problem is not mainly due to protein-protein or water-water interactions, whose parametrization varies the most between force fields and water models. In fact, as originally proposed in [ Best et al. J. Chem. Theory Comput. 2014, 10, 5113 - 5124.], balanced protein-water interactions may be the key to solving this problem. Our simulations using this approach produce results in very good agreement with experiment.