Interrelation between enzyme activity and stability with the local dynamics: Mutational and MD simulations based investigations
Dynamics plays crucial role in the function and stability of proteins. Earlier studies have provided ambivalent nature of these interrelations. Epistatic effects of amino acid substitutions on dynamics are an interesting strategy to investigate such relations. In this study we investigated the interrelation between dynamics with that of stability and activity of Bacillus subtilis lipase (BSL) using experimental and molecular dynamics simulation (MDS) approaches. Earlier we have identified many stabilising mutations in BSL using directed evolution. In this study these stabilizing mutations were clustered based on their proximity in the sequence into four groups (CM1 to 4). Activity, thermal stability, protease stability and aggregations studies were performed on these four mutants, along with the wild type BSL, to conclude that the mutations in each region contributed additively to the overall stability of the enzyme without suppressing the activity. Root mean square fluctuation and amide bond squared order parameter analysis from MDS revealed that dynamics has increased for CM1, CM2 and CM3 compared to the wild type in the amino acid region 105 to 112 and for CM4 in the amino acid region 22 to 30. In all the mutants core regions dynamics remained unaltered, while the dynamics in the rigid outer region (RMSF <0.05 nm) has increased. Alteration in dynamics, took place both in the vicinity (CM2, 0.41 nm) as well as far away from the mutations (CM1, 2.6 nm; CM3 1.5 nm; CM4 1.7 nm). Our data suggests that enhanced dynamics in certain regions in a protein may actually improve stability.\n\nStatement of SignificanceHow does a protein readjust its dynamics upon incorporation of an amino acid that improved its stability? Are the stabilizing effects of a substitution being local or non-local in nature? While there is an excellent documentation (from x-ray studies) of both local and non-local adjustments in interactions upon incorporation of a stabilizing mutations, the effect of these on the protein dynamics is less investigated. The stability and MD data presented here on four mutants, stabilized around four loop regions of a lipase, suggests that stabilizing effects of these mutations influence two specific regions leaving rest of the protein unperturbed. In addition, our data supports, observations by others, wherein enhancement in stability in a protein need not result in dampening of dynamics of a protein.