Massimiliano Anselmi

The Kinetics of Ligand Migration in Crystallized Myoglobin as Revealed by Molecular Dynamics Simulations

By using multiple molecular dynamics trajectories of photolyzed carbon monoxide (CO) within crystallized myoglobin, a quantitative description of CO diffusion and corresponding kinetics was obtained. Molecular dynamics results allowed us to construct a detailed kinetic model of the migration process, shedding light on the kinetic mechanism and relevant steps of CO migration and remarkably-well reproducing the available experimental data as provided by time-resolved Laue x-ray diffraction.

Theoretical modeling of vibroelectronic quantum states in complex molecular systems: Solvated carbon monoxide, a test case

In this paper we extend the perturbed matrix method by explicitly including the nuclear degrees of freedom, in order to treat quantum vibrational states in a perturbed molecule. In a previous paper we showed how to include, in a simple way, nuclear degrees of freedom for the calculation of molecular polarizability. In the present work we extend and generalize this approach to model vibroelectronic transitions, requiring a more sophisticated treatment.

Kinetics of Carbon Monoxide Migration and Binding in Solvated Neuroglobin As Revealed by Molecular Dynamics Simulations and Quantum Mechanical Calculations

Neuroglobin (Ngb) is a globular protein that reversibly binds small ligands at the six coordination position of the heme. With respect to other globins similar to myoglobin, Ngb displays some peculiarities as the topological reorganization of the internal cavities coupled to the sliding of the heme, or the binding of the endogenous distal histidine to the heme in the absence of an exogenous ligand. In this Article, by using multiple (independent) molecular dynamics trajectories (about 500 ns in total), the migration pathways of photolized carbon monoxide (CO) within solvated Ngb were analyzed, and a quantitative description of CO migration and corresponding kinetics was obtained. MD results, combined with quantum mechanical calculations on the CO-heme binding-unbinding reaction step in Ngb, allowed construction of a quantitative model representing the relevant steps of CO migration and rebinding.

Combining Crystallography and Molecular Dynamics: The Case of Schistosoma Mansoni Phospholipid Glutathione Peroxidase.

Oxidative stress is a widespread challenge for living organisms, and especially so for parasitic ones, given the fact that their hosts can produce reactive oxygen species (ROS) as a mechanism of defense. Thus, long lived parasites, such as the flatworm Schistosomes, have evolved refined enzymatic systems capable of detoxifying ROS. Among these, glutathione peroxidases (Gpx) are a family of sulfur or selenium‐dependent isozymes sharing the ability to reduce peroxides using the reducing equivalents provided by glutathione or possibly small proteins such as thioredoxin. As for other frontline antioxidant enzymatic systems, Gpxs are localized in the tegument of the Schistosomes, the outermost defense layer. In this article, we present the first crystal structure at 1.0 and 1.7 Å resolution of two recombinant SmGpxs, carrying the active site mutations Sec43Cys and Sec43Ser, respectively. The structures confirm that this enzyme belongs to the monomeric class 4 (phospholipid hydroperoxide) Gpx. In the case of the Sec to Cys mutant, the catalytic Cys residue is oxidized to sulfonic acid. By combining static crystallography with molecular dynamics simulations, we obtained insight into the substrate binding sites and the conformational changes relevant to catalysis, proposing a role for the unusual reactivity of the catalytic residue. Proteins 2010.

Dynamic Investigation of Protein Metal Active Sites: Interplay of XANES and Molecular Dynamics Simulations

The effect of structural disorder on the X-ray absorption near-edge structure (XANES) spectrum of a heme protein has been investigated using the dynamical description of the system derived from molecular dynamics (MD) simulations. The XANES spectra of neuroglobin (Ngb) and carbonmonoxy-neuroglobin (NgbCO) have been quantitatively reproduced, starting from the MD geometrical configurations, without carrying out any optimization in the structural parameter space. These results provide an important experimental validation of the reliability of the potentials used in the MD simulations and accordingly corroborate the consistency of the structural dynamic information on the metal center, related to its biological function. This analysis allowed us to demonstrate that the configurational disorder associated with the distortion of the heme plane and with the different orientations of the axial ligands can affect the XANES features at very low energy. Neglecting configurational disorder in the XANES quantitative analysis of heme proteins is a source of systematic errors in the determination of Fe coordination geometry. The combined use of XANES and MD is a novel strategy to enhance the resolution and reliability of the structural information obtained on metalloproteins, making the combination of these techniques powerful for metalloprotein investigations.

Molecular dynamics simulation of the neuroglobin crystal: comparison with the simulation in solution.

Neuroglobin (Ngb) is a monomeric protein that, despite the small sequence similarity with other globins, displays the typical globin fold. In the absence of exogenous ligands, the ferric and the ferrous forms of Ngb are both hexacoordinated to the distal and proximal histidines. In the ferrous form, oxygen, nitric oxide or carbon monoxide can displace the distal histidine, yielding a reversible adduct. Crystallographic data show that the binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities. Molecular dynamics (MD) simulations in solution show that the heme oscillates between two positions, much as the ones observed in the crystal structure, although the occupancy is different. The simulations also suggest that ligand binding in solution can affect the flexibility and conformation of residues connecting the C and D helices, referred to as the CD corner, which is coupled to the configuration adopted by the distal histidine. In this study, we report the results of 30 ns MD simulations of CO-bound Ngb in the crystal. Our goal was to compare the protein dynamical behavior in the crystal with the results supplied by the previous MD simulation of CO-bound Ngb in solution and the x-ray experimental data. The results show that the different environments (crystal or solution) affect the dynamics of the heme group and of the CD corner.

Analysis of infrared spectra of β-hairpin peptides as derived from molecular dynamics simulations.

Infrared temperature-dependent spectroscopy is a well-known tool to characterize folding/unfolding transitions in peptides and proteins, assuming that the higher the temperature, the higher the unfolded population. The infrared spectra at different temperatures of two β-hairpin peptides (gramicidin S analogues GS6 and GS10) are here reconstructed by means of molecular dynamics (MD) simulations and a theoretical-computational method based on the perturbed matrix method. The calculated temperature-dependent spectra result in good agreement with the experimental available spectra. The same methodology has been then used to reconstruct the spectra corresponding to the pure unfolded and folded states, as defined from the MD simulations, in order to better understand the temperature-dependent spectra and to help the interpretation of the experimental spectra. For example, our results show that in the case of the GS6 peptide the analysis of the temperature-dependent spectra cannot be used to investigate the folding/unfolding kinetics within the usual assumption that the higher the temperature, the higher the probability of the unfolded state.

Structure of the lipodepsipeptide syringomycin e in phospholipids and sodium dodecylsulphate micelle studied by circular dichroism, NMR spectroscopy and molecular dynamics

Syringomycin E (SRE) is a member of a family of lipodepsipeptides that characterize the secondary metabolism of the plant-associated bacteria Pseudomonas syringae pv. syringae. It displays phytotoxic, antifungal and haemolytic activities, due to the membrane interaction and ion channel formation. To gain an insight into the conformation of SRE in the membrane environment, we studied the conformation of SRE bound to SDS micelle, a suitable model for the membrane-bound SRE. In fact, highly similar circular dichroism (CD) spectra were obtained for SRE bound to sodium dodecylsulphate (SDS) and to a phospholipid bilayer, indicating the conformational equivalence of SRE in these two media, at difference with the CD spectrum of SRE in water solution. The structure of SDS-bound SRE was determined by NMR spectroscopy combined with molecular dynamics calculations in octane environment. The results of this study highlight the influence of the interaction with lipids in determining the three-dimensional structure of SRE and provide the basis for further investigations on structural determinants of syringomycin E-membrane interaction.

The effects of the L29F mutation on the ligand migration kinetics in crystallized myoglobin as revealed by molecular dynamics simulations

By using multiple molecular dynamics (MD) trajectories, a quantitative description of carbon monoxide (CO) migration within crystal of L29F myoglobin mutant (L29F‐Mb) was obtained. The aim was to provide a detailed model for ligand diffusion in the protein to be compared to the available L29F‐Mb experimental‐computational data and to the corresponding model kinetics we previously obtained for photolyzed CO within crystallized wild‐type myoglobin (wt‐Mb). Results suggest a clear migration pathway from distal pocket to the proximal site, similar to the one observed in wt‐Mb, with a relaxation kinetics differing from the wt‐Mb one essentially for the escape rate which is much higher in the mutant. Moreover MD data indicated a clear correlation between CO location within the protein and the conformation adopted by Phe29, well matching the available experimental data as obtained by time‐resolved X‐ray density maps. Such data, further validating the model used in the simulations, point out the subtle mutual effect between ligand diffusion and protein functional motions possibly explaining the observed dramatic variation of CO exit rate in L29F‐Mb. Proteins 2011.

Structural, Thermodynamic, and Kinetic Properties of Gramicidin Analogue GS6 Studied by Molecular Dynamics Simulations and Statistical Mechanics

Gramicidin S (GS) analogues belong to an important class of cyclic peptides, characterized by an antiparallel double‐stranded β‐sheet structure with Type II′ β‐turns. Such compounds can be used as model systems to understand the folding/unfolding process of β‐hairpins and more in general of β‐structures. In the present study, we specifically investigate the folding/unfolding behavior of the hexameric Gramicidin S analogue GS6 by using all‐atoms molecular dynamics (MD) simulations at different temperatures, coupled to a statistical mechanical model based on the Quasi Gaussian Entropy theory. Such an approach permits to describe the structural, thermodynamic, and kinetic properties of the peptide and to quantitatively characterize its folding/unfolding transitions.