RESUMO
We analyzed the thermal stability of the BstHPr protein through the site-directed point mutation Lys62 replaced by Ala residue using molecular dynamics simulations at five different temperatures: 298, 333, 362, 400, and 450 K, for periods of 1 µs and in triplicate. The results from the mutant thermophilic BstHPrm protein were compared with those of the wild-type thermophilic BstHPr protein and the mesophilic BsHPr protein. Structural and molecular interaction analyses show that proteins lose stability as temperature increases. Mutant and wild-type proteins behave similarly up to 362 K. However, at 400 K the mutant protein shows greater structural instability, losing more buried hydrogen bonds and exposing more of its non-polar residues to the solvent. Therefore, in this study, we confirmed that the salt bridge network of the Glu3-Lys62-Glu36 triad, made up of the Glu3-Lys62 and Glu36-Lys62 ion pairs, provides thermal stability to the thermophilic BstHPr protein.
Assuntos
Simulação de Dinâmica Molecular , Estabilidade Proteica , Ligação de Hidrogênio , Temperatura , Mutação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Substituição de Aminoácidos , Conformação Proteica , Mutagênese Sítio-DirigidaRESUMO
The histidine-containing phosphocarrier (HPr) is a monomeric protein conserved in Gram-positive bacteria, which may be of mesophilic or thermophilic nature. In particular, the HPr protein from the thermophilic organism B. stearothermophilus is a good model system for thermostability studies, since experimental data, such as crystal structure and thermal stability curves, are available. However, its unfolding mechanism at higher temperatures is yet unclear at a molecular level. Therefore, in this work, we researched the thermal stability of this protein using molecular dynamics simulations, subjecting it to five different temperatures during a time span of 1 µs. The analyses of the structural parameters and molecular interactions were compared with those of the mesophilic homologue HPr protein from B. subtilis. Each simulation was run in triplicate using identical conditions for both proteins. The results showed that the two proteins lose stability as the temperature increases, but the mesophilic structure is more affected. We found that the salt bridge network formed by the triad of Glu3-Lys62-Glu36 residues and the salt bridge made up of Asp79-Lys83 ion pair are key factors to keep stable the thermophilic protein, maintaining the hydrophobic core protected and the structure packed. In addition, these molecular interactions neutralize the negative surface charge, acting as "natural molecular staples".
Assuntos
Simulação de Dinâmica Molecular , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato , Estabilidade Enzimática , Proteínas de Bactérias/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/químicaRESUMO
Herein were tested 7 hydrophobic-polar sequences in two types of 2D-square space lattices, homogeneous and correlated, the latter simulating molecular crowding included as a geometric boundary restriction. Optimization of 2D structures was carried out using a variant of Dill's model, inspired by convex function, taking into account both hydrophobic (Dill's model) and polar interactions, including more structural information to reach better folding solutions. While using correlated networks, degrees of freedom in the folding of sequences were limited; as a result in all cases, more successful structural trials were found in comparison to a homogeneous lattice. The majority of employed sequences were designed by our workgroup, two of them were folded with other approaches, and another is a modified version of a previous sequence, initial forms of the other two have been employed but without taking into account polar-polar contributions. Three of them are newly proposed, intended to test the conjoint hydrophobic-hydrophobic and polar-polar contributions in crowded spaces. One sequence turned out to be the most difficult of the seven folded, this perhaps due to intrinsic (i) degrees of freedom and (ii) motifs of the expected 2D HP structure. Meanwhile two-sequence, although optimal folding was not achieved for neither of the two approaches, folding with correlated network approach not only produced better results than homogeneous space, but for them the best values found with crowding were very close to the expected optimal fitness. In general, five sequences were better folded with medium lattice units for correlated media; instead, another two sequences were better folded with a bit larger degree of lattice unit, revealing that depending on the degrees of freedom and particular folding, motifs in each sequence would require tuned crowding to achieve better folding. Therefore, the main goal herein was to obtain a modified 2D HP lattice model to mimic folding of proteins or secondary structures, like ß-sheets, taking into account both hydrophobic-hydrophobic and polar-polar interactions, and fold them in a crowded environment. This simple but enough construction would be conducted to determine the needed information to fold sequences in a sort of a minimal but complete heuristic model. Finally, we claim that all folded sequences into crowded spaces achieve better results than homogeneous ones.