Molecular Dynamics of Psychrophilic Enzyme Mutants Figure 5. Significant positive correlations between Ca atoms of domains A and B. The positive correlations are represented as grey sticks connecting the Ca atoms of the average structures from simulations. Grey spheres represent the coordinated ions. The regions of loop 7 and 8 are highlighted by circles in AHA and PPA, respectively. doi:10.1371/journal.pone.0024214.g005 interconnecting L7 (Figure 5, Figure 8A–B) and by an emerging higher flexibility, ‘‘mesophilic-like’’, in the region of L8 loop (Figure 4) in some mutant variants. A detailed analysis of all the pairs of residues with cross- correlated motions, which are modified in AHA with respect to PPA or AHA mutants has been carried out, in particular focusing on the residues which differ in AHA and PPA, as derived by their structural alignment. In particular, a stable network of correlations connecting residues D264A (which belongs to the catalytic triad), V260A, D261A, N262A, V275A, T277A, F278A and K334A in AHA (Figure 8A), is lost in the AHA mutant variants, in parallel with a local modification of the electrostatic interactions involving K334A. K334A in AHA cooperates, by salt bridges with E336A and E279A and an aromatic interaction with F278A, to determine the correlated motions in this region and the dynamic properties of AHA L7 loop. On the contrary in PPA, only the counterparts of E336A (E385P) and F278A (F315P) are present, whereas K334A, E279A are replaced by V383P and W316P. Moreover, along with these differences, I367P (which is located in the L8 insertion typical of mesophilic amylases and absent in AHA) in cooperation with the salt bridge network R343P-D381P-K368P (Figure 6 and 7), plays a crucial role as critical residue in promoting a cluster of PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24214