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The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric and filamentous actin. Recent work on the protein Actophorin from the amoeba Acanthamoeba castellani identified a series of site directed mutations that increase the thermal stability of the protein by 22 ◦C. Here, we expand this observation by showing that the mutant protein is also significantly stable to both equilibrium and kinetic chemical denaturation, and employ computer simulations to account for the increase in thermal or chemical stability through an accounting of atomic-level interactions. Specifically, the potential of mean force (PMF) can be obtained from steered molecular dynamics (SMD) simulations in which a protein is unfolded. However, SMD can be inefficient for large proteins as they require large solvent boxes, and computationally expensive as they require increasingly many SMD trajectories to converge the PMF. Adaptive steered molecular dynamics (ASMD) overcomes the second of these limitations by steering the particle in stages, which allows for convergence of the PMF using fewer trajectories compared to SMD. Use of the telescoping water scheme within ASMD partially overcomes the first of these limitations by reducing the number of waters at each stage to only those needed to solvate the structure within a given stage. In the PMFs obtained from ASMD, the work of unfolding Acto-2 was found to be higher than the Acto-WT by approximately 120 kCal/mol and reflects the increased stability seen in the chemical denaturation experiments. The evolution of the average number of hydrogen bonds andnumber of salt bridges during the pulling process provides a mechanistic view of the structural changes of the Actophorin protein as it is unfolded, and how it is affected by the mutation in concert with the energetics reported through the PMF. 

Abstract As the epidemic of single‐use plastic worsens, it has become critical to identify fully renewable plastics such as those that can be degraded using enzymes. Here we describe the structure and biochemistry of an alkaline poly[(R)‐3‐hydroxybutyric acid] (PHB) depolymerase from the soil thermophileLihuaxuella thermophila. Like other PHB depolymerases or PHBases, theLihuaxuellaenzyme is active against several different polyhydroxyalkanoates, including homo‐ and heteropolymers, butL. thermophilaPHB depolymerase (LtPHBase) is unique in that it also hydrolyzes polylactic acid and polycaprolactone.LtPHBase exhibits optimal activity at 70°C, and retains 88% of activity upon incubation at 65°C for 3 days. The 1.2 Å resolution crystal structure reveals an α/β‐hydrolase fold typical of PHBases, but with a shallow active site containing the catalytic Ser‐His‐Asp‐triad that appears poised for broad substrate specificity.LtPHBase holds promise for the depolymerization of PHB and related bioplastics at high temperature, as would be required in bioindustrial operations like recycling or landfill management. 

Abstract The potentials of mean force (PMFs) along the end‐to‐end distance of two different helical peptides have been obtained and benchmarked using the adaptive steered molecular dynamics (ASMD) method. The results depend strongly on the choice of force field driving the underlying all‐atom molecular dynamics, and are reported with respect to the three most popular CHARMM force field versions: c22, c27 and c36. Two small peptides,and 1PEF, serve as the particular case studies. The comparisons between the versions of the CHARMM force fields provides both a qualitative and quantitative look at their performance in forced unfolding simulations in which peptides undergo large changes in structural conformations. We find that ASMD with the underlying c36 force field provides the most robust results for the selected benchmark peptides.