Molecular Dynamics of Psychrophilic Enzyme Mutants Figure 8. Focus on cross-correlated residues in L7–L8 region and summary of proposed residues for mutagenesis studies. A. The cross-correlation network of AHA which is lost in PPA and AHA mutants (AHA numbering) in the proximity of L7/L8 region. B. The cross-correlation network of PPA mediated by I367P not restorable in AHA mutants (PPA numbering). C. The cross-correlation network of AHA which is lost in PPA and AHA mutants (AHA numbering) in the proximity of L3/L5 loops. The positive correlations represented as red sticks connecting the Ca atoms. D. The hub residues in promoting AHA and PPA characteristic dynamic patterns, not effectively modified in the mutants and which can be a suitable residue subset to be experimentally investigated are mapped on the average 3D structures from AHA (white) and PPA (black) simulations in magenta (AHA) and blue (PPA), respectively. doi:10.1371/journal.pone.0024214.g008 mutants, a combination of local and long range effects clearly modifies the interactions and the dynamic patterns of K334A, favoring interactions between K334A with the aforementioned D306A (Figure 6 and 7) in AHA (corresponding to R343P residue involved in the PPA salt bridge network), and, in turn changed the local dynamic pattern toward a ‘‘mesophilic-like’’ behavior. The mutations affecting K334 properties also alter the composition of the most populated cluster of electrostatic interactions typical of AHA (Figure 6 and 7; Figure S7 in Supporting Information S1). In particular, in the AHA mutants, this important salt-bridge cluster is divided into two smaller and non-communicating clusters, influencing dynamic and structural properties of the surrounding regions in the proximity of the catalytic site and including the L7/ L8 regions. It is worth to mention that the effects induced by the AHA mutations are still not completely comparable to the L8 loop flexibility pattern of PPA and its cross-correlation map. This result is not surprising considering the different length and composition of L8 loop in PPA, suggesting that a mesophilic dynamic fingerprint could be more successfully acquired combining some of the investigated AHA mutations, with other mutations or amino acid insertion typical of PPA L8 loop. In fact, integrating the data collected from the different analyses of the MD trajectories, it clearly emerged that the main determinant of differences in cross- correlated motions and flexibility in L7/L8 region in AHA mutant variants relies on a subset of residues with a pivotal role, which are suitable candidates for further mutagenesis experiments, with particular attention to the introduction of a residue corresponding to I367P. However, it is striking to observe that AHA mutants manage to attain flexibility patterns similar to that of the mesophilic homolog, especially because all mutations are located far from this region, highlighting the ability of single amino-acidic changes to signifi- cantly modulate the flexibility of distal regions of AHA toward ‘‘mesophilic-like’’ properties. Modification in dynamic properties in proximity of L3 and L5 loops Domain B is the longest unstructured region in both AHA and PPA (44 and 67 residues long, respectively) and connects b3 to a3 (Figure 1), covering one side of the central barrel and closing the substrate-binding cleft. Domain B interacts with loop b52a5 (L5), which in part (residues 200–210) protrudes towards the active site. A hydrophobic cluster between a3, loop a32b4, b4 and loop a42b5 is reinforced by mutations Q164I and V196F (Figure 3), while mutation N150D allows the establishment of the D150- K190 salt bridge between a3 and a4 (Figure 7). Accordingly, mutants bearing all three these mutations (AHA5 and AHA5SS) show the lowest flexibility in the L5 region (Figure 3 and 4). V196F mutation seems to have a prominent role, since it is capable, alone, of yielding a rigidification similar to that of the multiple mutants (Figure 3 and 4). However, the engineered mutations in AHA do not completely affect the pattern of flexibility of AHA L3 and L5 regions. In PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24214