Genome sequence of a diabetes-prone rodent reveals a mutation hotspot around the ParaHox gene cluster.

The sand rat Psammomys obesus is a gerbil species native to deserts of North Africa and the Middle East, and is constrained in its ecology because high carbohydrate diets induce obesity and type II diabetes that, in extreme cases, can lead to pancreatic failure and death. We report the sequencing of the sand rat genome and discovery of an unusual, extensive, and mutationally biased GC-rich genomic domain. This highly divergent genomic region encompasses several functionally essential genes, and spans the ParaHox cluster which includes the insulin-regulating homeobox gene Pdx1. The sequence of sand rat Pdx1 has been grossly affected by GC-biased mutation, leading to the highest divergence observed for this gene across the Bilateria. In addition to genomic insights into restricted caloric intake in a desert species, the discovery of a localized chromosomal region subject to elevated mutation suggests that mutational heterogeneity within genomes could influence the course of evolution.

Molecular Effects of Concentrated Solutes on Protein Hydration, Dynamics, and Electrostatics.

Most studies of protein structure and function are performed in dilute conditions, but proteins typically experience high solute concentrations in their physiological scenarios and biotechnological applications. High solute concentrations have well-known effects on coarse protein traits like stability, diffusion, and shape, but likely also perturb other traits through finer effects pertinent at the residue and atomic levels. Here, NMR and molecular dynamics investigations on ubiquitin disclose variable interactions with concentrated solutes that lead to localized perturbations of the protein's surface, hydration, electrostatics, and dynamics, all dependent on solute size and chemical properties. Most strikingly, small polar uncharged molecules are sticky on the protein surface, whereas charged small molecules are not, but the latter still perturb the internal protein electrostatics as they diffuse nearby. Meanwhile, interactions with macromolecular crowders are favored mainly through hydrophobic, but not through polar, surface patches. All the tested small solutes strongly slow down water exchange at the protein surface, whereas macromolecular crowders do not exert such strong perturbation. Finally, molecular dynamics simulations predict that unspecific interactions slow down microsecond- to millisecond-timescale protein dynamics despite having only mild effects on pico- to nanosecond fluctuations as corroborated by NMR. We discuss our results in the light of recent advances in understanding proteins inside living cells, focusing on the physical chemistry of quinary structure and cellular organization, and we reinforce the idea that proteins should be studied in native-like media to achieve a faithful description of their function.

Structure-function paradigm in human myoglobin: how a single-residue substitution affects NO reactivity at low pO2.

This work is focused on the two more expressed human myoglobin isoforms. In the literature, their different overexpression in high-altitude natives was proposed to be related to alternative/complementary functions in hypoxia. Interestingly, they differ only at residue-54, lysine or glutamate, which is external and far from the main binding site. In order to ascertain whether these two almost identical myoglobins might exert different functions and to contribute to a deeper understanding about myoglobin's oxygen-level dependent functioning, they have been compared with respect to dynamics, heme electronic structure, and NO reactivity at different O2 levels. Electron paramagnetic resonance (EPR) spectroscopy was employed to investigate the electronic structure of the nitrosyl-form, obtaining fundamental clues about a different bond interaction between the heme-iron and the proximal histidine and highlighting striking differences in NO reactivity, especially at a very low pO2. The experimental results well matched with the information provided by molecular dynamics simulations, which showed a significantly different dynamics for the two proteins only in the absence of O2. The single mutation differentiating the two myoglobins resulted in strongly affecting the plasticity of the CD-region (C-helix-loop-D-helix), whose fluctuations, being coupled to the solvent, were found to be correlated with the dynamics of the distal binding site. In the absence of O2, on the one hand a significantly different probability for the histidine-gate opening has been shown by MD simulations, and on the other a different yield of myoglobin-NO formation was experimentally observed through EPR.

All-atom simulations of crowding effects on ubiquitin dynamics.

It is well-known that crowded environments affect the stability of proteins, with strong biological and biotechnological implications; however, beyond this, crowding is also expected to affect the dynamic properties of proteins, an idea that is hard to probe experimentally. Here we report on a simulation study aimed at evaluating the effects of crowding on internal protein dynamics, based on fully all-atom descriptions of the protein, the solvent and the crowder. Our model system consists of ubiquitin, a protein whose dynamic features are closely related to its ability to bind to multiple partners, in a 325 g L⁻¹ solution of glucose in water, a condition widely employed in in vitro studies of crowding effects. We observe a slight reduction in loop flexibility accompanied by a dramatic restriction of the conformational space explored in the timescale of the simulations (∼0.5 µs), indicating that crowding slows down collective motions and the rate of exploration of the conformational space. This effect is attributed to the extensive and long-lasting interactions observed between protein residues and glucose molecules throughout the entire protein surface. Potential implications of the observed effects are discussed.

Facilitated permeation of antibiotics across membrane channels--interaction of the quinolone moxifloxacin with the OmpF channel.

The facilitated influx of moxifloxacin through the most abundant channel in the outer cell wall of gram-negative bacteria was investigated. Molecular modeling provided atomic details of the interaction with the channel surface, revealed the preferred orientation of the antibiotic along its pathway, and gave an estimated time necessary for translocation. High-resolution conductance measurements on single OmpF trimers allowed the passages of individual moxifloxacin molecules to be counted. The average mean residence time of 50 micros is in agreement with the predicted strong interaction from the modeling. In contrast, control measurements with nalidixic acid, a hydrophobic antibiotic that rather permeates across the lipid membrane, revealed a negligible interaction. The spectral overlap of tryptophan with moxifloxacin was suitable for a FRET study of the protein-antibiotic interaction. Combining molecular dynamics simulations with selective quenching identified an interaction of moxifloxacin with Trp61 inside the OmpF channel, whereas nalidixic acid showed preferential interaction with Trp214 on the channel exterior. An understanding of the detailed molecular interactions between the antibiotic and its preferred channel may be used to develop new antibiotics with improved uptake kinetics.

Probing the early stages of prion protein (PrP) aggregation with atomistic molecular dynamics simulations.

Prions are self-replicating infectious proteinaceous agents whose conformations are capable of forming amyloid-like aggregate fibrils. Here we present molecular dynamics simulations aimed at investigating the aggregation process of the ß-rich H2H3 domain of the ovine prion protein (H2H3-OvPrPSc), known to be the portion of prion protein carrying oligomerization activity.

Assembly of Influenza Hemagglutinin Fusion Peptides in a Phospholipid Bilayer by Coarse-grained Computer Simulations.

Membrane fusion is critical to eukaryotic cellular function and crucial to the entry of enveloped viruses such as influenza and human immunodeficiency virus. Influenza viral entry in the host cell is mediated by a 20-23 amino acid long sequence, called the fusion peptide (FP). Recently, possible structures for the fusion peptide (ranging from an inverted V shaped α-helical structure to an α-helical hairpin, or to a complete α-helix) and their implication in the membrane fusion initiation have been proposed. Despite the large number of studies devoted to the structure of the FP, the mechanism of action of this peptide remains unclear with several mechanisms having been suggested, including the induction of local disorder, promoting membrane curvature, and/or altering local membrane composition. In recent years, several research groups have employed atomistic and/or coarse-grained molecular dynamics (MD) simulations to investigate the matter. In all previous works, the behavior of a single FP monomer was studied, while in this manuscript, we use a simplified model of a tripeptide (TP) monomer of FP (TFP) instead of a single FP monomer because each Influenza Hemagglutinin contains three FP molecules in the biological system. In this manuscript we report findings targeted at understanding the fusogenic properties and the collective behavior of these trimers of FP peptides on a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine model membrane. Here we show how the TFP monomers self-assemble into differently sized oligomers in the presence of the membrane. We measure the perturbation to the structure of the phospholipid membrane caused by the presence of these TFP oligomers. Our work (i) shows how self-assembly of TFP in the presence of the membrane induces non negligible deformation to the membrane and (ii) could be a useful starting point to stimulate discussion and further work targeted to fusion pore formation.

New strategies for integrative dynamic modeling of macromolecular assembly.

Data reporting on structure and dynamics of cellular constituents are growing with increasing pace enabling, as never before, the understanding of fine mechanistic aspects of biological systems and providing the possibility to affect them in controlled ways. Nonetheless, experimental techniques do not yet allow for an arbitrary level of resolution on cellular processes in situ. By consistently integrating a variety of diverse experimental data, molecular modeling is optimally poised to enhance to near-atomistic resolution our understanding of molecular recognition in large assemblies. Within this integrative modeling context, we briefly review in this chapter the recent progresses of molecular simulations at the atomistic and coarse-grained level of resolution to explore protein-protein interactions. In particular, we discuss our recent contributions in this field, which aim at providing a robust bridge between novel optimization algorithms and multiscale molecular simulations for a consistent integration of experimental inputs. We expect that, with the ever-growing sampling ability of molecular simulations and the tireless progress of experimental methods, the impact of such dynamic-based approach could only be more effective with time, contributing to provide detailed description of cellular organization.

Dissecting the effects of concentrated carbohydrate solutions on protein diffusion, hydration, and internal dynamics.

We present herein a thorough description of the effects of high glucose concentrations on the diffusion, hydration and internal dynamics of ubiquitin, as predicted from extensive molecular dynamics simulations on several systems described at fully atomistic level. We observe that the protein acts as a seed that speeds up the natural propensity of glucose to cluster at high concentration; the sugar molecules thus aggregate around the protein trapping it inside a dynamic cage. This process extensively dehydrates the protein surface, restricts the motions of the remaining water molecules, and drags the large-scale, collective motions of protein atoms slowing down the rate of exploration of the conformational space despite only a slight dampening of fast, local dynamics. We discuss how these effects could be relevant to the function of sugars as preservation agents in biological materials, and how crowding by small sticky molecules could modulate proteins across different reaction coordinates inside the cellular cytosol.

Electrostatic-Consistent Coarse-Grained Potentials for Molecular Simulations of Proteins.

We present a new generation of coarse-grained (CG) potentials that account for a simplified electrostatic description of soluble proteins. The treatment of permanent electrostatic dipoles of the backbone and polar side-chains allows to simulate proteins, preserving an excellent structural and dynamic agreement with respective reference structures and all-atom molecular dynamics simulations. Moreover, multiprotein complexes can be well described maintaining their molecular interfaces thanks to the ability of this scheme to better describe the actual electrostatics at a CG level of resolution. An efficient and robust heuristic algorithm based on particle swarm optimization is used for the derivation of CG parameters via a force-matching procedure. The ability of this protocol to deal with high dimensional search spaces suggests that the extension of this optimization procedure to larger data sets may lead to the generation of a fully transferable CG force field. At the present stage, these electrostatic-consistent CG potentials are easily and efficiently parametrized, show a good degree of transferability, and can be used to simulate soluble proteins or, more interestingly, large macromolecular assemblies for which long all-atom simulations may not be easily affordable.

Molecular basis of enrofloxacin translocation through OmpF, an outer membrane channel of Escherichia coli--when binding does not imply translocation.

The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site, which facilitates translocation. Fluorescence steady-state measurements agree with the different location of binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation.