Effect of the Lys62Ala Mutation on the Thermal Stability of BstHPr Protein by Molecular Dynamics.

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.

The Impact of Residual Dispersant on the Flocculation and Sedimentation of Synthetic Tailings in Seawater.

Dispersants under certain conditions favor the flotation of molybdenite in seawater; however, it is not clear if the entrainment of residues to the thickening stage can compromise the quality of the clarified water. In this work, the impact of small concentrations of sodium hexametaphosphate (SHMP) on the flocculation and sedimentation of synthetic tailings containing kaolinite, muscovite, and quartz in seawater is evaluated. The flocculant polymer is a high-molecular-weight polyacrylamide, and the pH is alkaline. The results are auspicious for mineral processing. On the one hand, the impact of SHMP is not entirely negative and can be lessened by limiting entrainment, which is good for copper and molybdenum ore processing. On the other hand, if the small increase in turbidity generated by the SHMP is tolerated, it is possible to expect improved settling speeds. Without SHMP, large but light agglomerates are formed. With SHMP, smaller but denser aggregates are formed, settling faster, and minute aggregates increase turbidity. The underlying mechanism derives from the competition between SHMP and polymer chains for the cations in solution; the result is a greater repulsion between the chains, which leads to greater repulsion and thus dispersion of smaller flocculant coils. The study shows that SHMP in concentrations of 1 to 3 kg/t is perfectly acceptable. The results represent an advance in the understanding of SHMP interactions with polymers and minerals in water clarification, which should be of interest to the industry whose sustainability in some regions depends on closing the water cycle.

An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity.

Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin.IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

Dynamical study, hydrogen bond analysis, and constant pH simulations of the beta carbonic anhydrase of Methanobacterium thermoautotrophicum.

Within the five classes (α, ß, γ, δ, and ζ) of carbonic anhydrases (CAs) the first two, containing mammal and plant representatives, are the most studied among all CAs. In this study, we have focused our investigation on the beta-class carbonic anhydrase of Methanobacterium thermoautotrophicum. We investigated both the importance of the Asp-Arg dyad near the catalytic zinc-bound water and the possible roles that water molecules within the active site and residues near the entrance of the catalytic cleft have on the first step of the enzyme's reaction mechanism. Hydrogen-bonding analysis of selected residues within the active site and constant pH replica exchange molecular dynamics constant pH replica exchange simulations were performed. The latter was done in order to evaluate the pKa values of possible proton acceptors. We found an intricate hydrogen-bonding network involving two acidic residues within the active site, Asp16 and Asp34, and the catalytic water molecule. We also observed a very strong interaction between the zinc-bound water and residues Asp34 and Arg36. This interaction was not significantly affected by the change in the protonation state of both the catalytic water and aspartate residue 34. The pKa analysis show that the effect of the R36A mutation affects not only the possible proton acceptors, but also the catalytic water itself.