The Role of Protein Structural Ensembles in Thermostability and Ligand Binding

The role of protein structural ensembles has been shown to be very important for different physical and chemical properties of proteins. The work presented in this dissertation explores two of these properties:i) Thermostability, by characterizing, at three different temperatures, the dynamics of aminoglycoside nucleotidyltransferase 4’ (ANT). This homodimeric enzyme detoxifies antibiotics. It possess two known variants, D80Y and T130K, with higher melting temperatures than the wild type. These mutations, however, would cause changes in the distributions of conformations in the ensemble and, consequently, on the dynamics of the protein. To test this hypothesis, the wild type and variants were examined by using molecular dynamics simulations and the results were compared with previous experimental information in order to characterize the similarities and differences between the, so-called, thermophilic and thermostable variants of this enzyme.ii) Ligand binding: Since proteins are in general dynamic structures, it would be expected that the effectiveness of ligand binding varies as the protein’s conformation changes. One of the most targeted protein family in the field of drug discovery/design is the G-Protein Coupled Receptor (GPCR) family. Over 30% of approved drugs target this family of proteins. This project examines, via in silico experiments, the differences in ligand binding between different conformations of GPCRs. To this end, GPCR ligand structures, actual binding (actives) and non-binding (decoys) ligands, were obtained from public databases, and eight GPCRs structures were selected to generate 5,000 conformational states for each protein. Ensemble-based docking was performed on representative structures of these 5,000 conformers and on a subset of 3,000 conformers from each of the eight proteins. Decoys and statistical analysis were incorporated in the docking simulations to test whether the sampled protein conformations can bind active ligands in greater numbers than the random selection from the pool of active and decoys. The results show that some conformations bind more ligands than other conformations, random selection, or the crystal structure. Characterizing the entire ensemble of protein conformations can improve the number of bound active ligands identified computationally, compared to random selection of compounds or docking using only a single crystal structure

Revisiting the Sweet Taste Receptor T1R2-T1R3 through Molecular Dynamics Simulations Coupled with a Noncovalent Interactions Analysis

It is nowadays widely accepted that sweet taste perception is elicited by the activation of the heterodimeric complex T1R2-T1R3, customarily known as sweet taste receptor (STR). However, the interplay between STR and sweeteners has not yet been fully clarified. Here through a methodology coupling molecular dynamics and the independent gradient model (igm) approach we determine the main interacting signatures of the closed (active) conformation of the T1R2 Venus flytrap domain (VFD) toward aspartame. The igm methodology provides a rapid and reliable quantification of noncovalent interactions through a score (Δginter) based on the attenuation of the electronic density gradient when two molecular fragments approach each other. Herein, this approach is coupled to a 100 ns molecular dynamics simulation (MD-igm) to explore the ligand-cavity contacts on a per-residue basis as well as a series of key inter-residue interactions that stabilize the closed form of VFD. We also apply an atomic decomposition scheme of noncovalent interactions to quantify the contribution of the ligand segments to the noncovalent interplay. Finally, a series of structural modification on aspartame are conducted in order to obtain guidelines for the rational design of novel sweeteners. Given that innovative methodologies to reliably quantify the extent of ligand-protein coupling are strongly demanded, this approach combining a noncovalent analysis and MD simulations represents a valuable contribution, that can be easily applied to other relevant biomolecular systems.Revisión por pare