Calculation of Protein Folding Thermodynamics Using Molecular Dynamics Simulations.

Despite advances in artificial intelligence methods, protein folding remains in many ways an enigma to be solved. Accurate computation of protein folding energetics could help drive fields such as protein and drug design and genetic interpretation. However, the challenge of calculating the state functions governing protein folding from first-principles remains unaddressed. We present here a simple approach that allows us to accurately calculate the energetics of protein folding. It is based on computing the energy of the folded and unfolded states at different temperatures using molecular dynamics simulations. From this, two essential quantities (ΔH and ΔCp) are obtained and used to calculate the conformational stability of the protein (ΔG). With this approach, we have successfully calculated the energetics of two- and three-state proteins, representatives of the major structural classes, as well as small stability differences (ΔΔG) due to changes in solution conditions or variations in an amino acid residue.

Molecular dynamics simulations for genetic interpretation in protein coding regions: where we are, where to go and when.

The increasing ease with which massive genetic information can be obtained from patients or healthy individuals has stimulated the development of interpretive bioinformatics tools as aids in clinical practice. Most such tools analyze evolutionary information and simple physical-chemical properties to predict whether replacement of one amino acid residue with another will be tolerated or cause disease. Those approaches achieve up to 80-85% accuracy as binary classifiers (neutral/pathogenic). As such accuracy is insufficient for medical decision to be based on, and it does not appear to be increasing, more precise methods, such as full-atom molecular dynamics (MD) simulations in explicit solvent, are also discussed. Then, to describe the goal of interpreting human genetic variations at large scale through MD simulations, we restrictively refer to all possible protein variants carrying single-amino-acid substitutions arising from single-nucleotide variations as the human variome. We calculate its size and develop a simple model that allows calculating the simulation time needed to have a 0.99 probability of observing unfolding events of any unstable variant. The knowledge of that time enables performing a binary classification of the variants (stable-potentially neutral/unstable-pathogenic). Our model indicates that the human variome cannot be simulated with present computing capabilities. However, if they continue to increase as per Moore's law, it could be simulated (at 65°C) spending only 3 years in the task if we started in 2031. The simulation of individual protein variomes is achievable in short times starting at present. International coordination seems appropriate to embark upon massive MD simulations of protein variants.

Stereoselective synthesis and biological evaluation as inhibitors of hepatitis C virus RNA polymerase of GSK3082 analogues with structural diversity at the 5-position.

GSK3082 - a hepatitis C virus RNA polymerase inhibitor - and a series of analogues with structural diversity at the 5-position were prepared from a 2,2,4,5-tetrasubstituted pyrrolidine obtained with a well-defined stereochemistry from the 1,3-dipolar cycloaddition of the chiral imino ester derived from leucine tert-butyl ester and (R)-2,3-O-isopropylideneglyceraldehyde with methyl acrylate. The chiral 2,2-dimethyl-1,3-dioxolane moiety provided by the glyceraldehyde served as a synthetic equivalent for different substituents and functional groups and these transformations usually required mild reaction conditions and simple work-up procedures. The inhibitory activity of the resulting GSK3082 analogues was studied in vitro in a cell-based assay of the subgenomic HCV RNA replication system. Some of the analogues showed good inhibitory activity with IC50 values in the nanomolar concentration range.

A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aß1-42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms.

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer's disease, Parkinson's disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aß1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aß1-42, α-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them. Graphical abstract ᅟ.

Direct examination of the relevance for folding, binding and electron transfer of a conserved protein folding intermediate.

Near the minimum free energy basin of proteins where the native ensemble resides, partly unfolded conformations of slightly higher energy can be significantly populated under native conditions. It has been speculated that they play roles in molecular recognition and catalysis, but they might represent contemporary features of the evolutionary process without functional relevance. Obtaining conclusive evidence on these alternatives is difficult because it requires comparing the performance of a given protein when populating and when not populating one such intermediate, in otherwise identical conditions. Wild type apoflavodoxin populates under native conditions a partly unfolded conformation (10% of molecules) whose unstructured region includes the binding sites for the FMN cofactor and for redox partner proteins. We recently engineered a thermostable variant where the intermediate is no longer detectable. Using the wild type and variant, we assess the relevance of the intermediate comparing folding kinetics, cofactor binding kinetics, cofactor affinity, X-ray structure, intrinsic dynamics, redox potential of the apoflavodoxin-cofactor complex (Fld), its affinity for partner protein FNR, and electron transfer rate within the Fld/FNR physiological complex. Our data strongly suggest the intermediate state, conserved in long-chain apoflavodoxins, is not required for the correct assembly of flavodoxin nor does it contribute to shape its electron transfer properties. This analysis can be applied to evaluate other native basin intermediates.

The mechanism of water/ion exchange at a protein surface: a weakly bound chloride in Helicobacter pylori apoflavodoxin.

Binding/unbinding of small ligands, such as ions, to/from proteins influences biochemical processes such as protein folding, enzyme catalysis or protein/ligand recognition. We have investigated the mechanism of chloride/water exchange at a protein surface (that of the apoflavodoxin from Helicobacter pylori) using classical all-atom molecular dynamics simulations. They reveal a variety of chloride exit routes and residence times; the latter is related to specific coordination modes of the anion. The role of solvent molecules in the mechanism of chloride unbinding has been studied in detail. We see no temporary increase in chloride coordination along the release process. Instead, the coordination of new water molecules takes place in most cases after the chloride/protein atom release event has begun. Moreover, the distribution function of water entrance events into the first chloride solvation shell peaks after chloride protein atom dissociation events. All these observations together seem to indicate that water molecules simply fill the vacancies left by the previously coordinating protein residues. We thus propose a step-by-step dissociation pathway in which protein/chloride interactions gradually break down before new water molecules progressively fill the vacant positions left by protein atoms. As observed for other systems, water molecules associated with bound chloride or with protein atoms have longer residence times than those bound to the free anion. The implications of the exchange mechanism proposed for the binding of the FMN (Flavin Mononucleotide) protein cofactor are discussed.