residues, which also interact with the protein N-terminal extremity (residues 1–13P); whereas in AHA the N-terminal is 8 residues shorter, thus not allowing the extension of the network (Figure S5A in Supporting Information S1). Moreover, the presence of two arginines (R92P and R252P) in the macro-aromatic cluster of PPA, and not in AHA, accounts for a further stabilizing contribution due to cation-PI interactions. AHA mutants bearing V196F (AHAVF, AHA5SS and AHA5) show a local increase in the surrounding hydrophobicity (Figure 3), thanks to the favorable packing induced by mutation, even in absence of the reconstruc- tion of other more complex interactions. T232V mutation strengthens a hydrophobic cluster between a6 and a6-b7, in the proximity of the interface between domains A and C (Figure 1, Figure S5B–C in Supporting Information S1). AHA variants bearing T232V mutation (AHATV, AHA5SS and AHASS) show a higher persistence of hydrophobic residues and a consequent increase in surrounding hydrophobicity (Figure 3), including in particular L238A, F244A and W248A (Figure S5C in Supporting Information S1). K300R mutation, in the chloride-binding site and in the proximity of the active site (Figure 1), should provide bi-dentate coordination for the Cl2 ion, even if a more complex network of interactions is mediated by R337P in PPA (Figure S5D and S6 in Supporting Information S1). In fact, the mutation is located among three macro-clusters of aromatic residues, conserved in both AHA and PPA (Figure S6 in Supporting Information S1). The K300R mutation has slight effects on the persistence of the aromatic residues in the surrounding of the mutated residues (Figure S5D in Supporting Information S1), while it strengthens a salt bridge with D261 (R337P-D297P in PPA). This is in line with the observed role of arginine if compared to lysine in an aromatic context [40], role which is not ascribable to a intrinsic higher cation-PI binding ability of Arg. In fact, the Arg side chain is larger and less water-solvated than the cognate Lys thus, likely to benefit from a better van der Waals interactions with aromatic rings. Moreover, as suggested also by Thornton and colleagues [41], the Arg side chain can still be involved in H-bonds or salt bridges while simultaneously interacting with aromatic rings, whereas Lys typically has to relinquish H-bonds to bind to aromatic residues. Effects of the mutation on the structural and dynamic properties of AHA To achieve an overall description of the structural and dynamic properties of the AHA mutants, several properties have been analyzed, from H-bonds and surrounding hydrophobicity (Hp) (Figure 2 and 3), rmsf profiles (Figure 3), the anisotropic temperature factors of the essential subspace (Figure 4), most relevant positive correlated motions (Figure 5), and salt bridge interactions and networks (Figure 6, 7, Figure S7 and Table S1 in Supporting Information S1), along with the clusters of salt-bridges defined by a spatial proximity criterion (Figure 6, 7, Figure S7 and Table S1 in Supporting Information S1). In fact, it is well-known that the protein stability results from a delicate balance between different weak intramolecular interac- tions, which in turn also modulate protein dynamics. Electrostatic interactions, and in particular salt-bridges, have been shown to play a crucial role in protein stability [42–45], featuring both local and distal variable effects [46,47]. Salt-bridges are generally highly flexible and cooperatively organized in salt-bridge networks in the protein structure, strongly influencing protein dynamics. Therefore, they are a suitable group of intramolecular interactions which may be used as a reference to identify dynamic intramolecular networks and how they are modified by mutations. In light of these observation, we define not Molecular Dynamics of Psychrophilic Enzyme Mutants only salt-bridge pairs and networks, according to their persistence during the simulations, but also how they are organized in spatial proximity clusters, as previously applied to other comparative studies on extremophilic enzymes [48]. Our description does not account for calculations of salt-bridge strength and its influence on protein stability [46,47,49], which still lacks an accurate definition in extremophilic a-amylases, both since experiments and simula- tions at different temperatures are still not available and also since most of the residues involved in salt-bridges which differ between AHA and AHA mutants are generally solvent exposed (with a solvent accessibility of their side chains generally higher than 30% during the simulation time) and therefore likely not to be negatively influenced by desolvation penalties. Globally, few differences, mainly in domain A, are observed in terms of H-bond content and surrounding hydrophobicity (Figure 2 and 3). All mutants show a reduced content of residues with low surrounding hydrophobicity, an increase in residues characterized by higher Hp values, especially the variants which include hydro- phobic or aromatic mutations (AHA5SS, AHA5, AHATV, AH AQI, AHAVF) (Figure 2 and 3), as also discussed above. The patterns of correlated motions and flexibility of AHA and PPA present distinctive features, which have been described in details in a previous publication [50]. The main relevant aspect is related to ba loops of different length and composition in the surroundings of the active site. In particular, AHA has most of its flexibility localized on the L7 loop (b7-a7) and the C-terminal extremity of L3 (L3C), as well as at the L3–L5 interface, whereas PPA has the highest flexibility scattered far from the catalytic site toward the most solvent-exposed L8 and the N-terminal of L3 (L3N) (Figure 3 and 4). Interestingly, in AHA mutants, flexibility (Figure 4) and correlation patterns (Figure 5) typical of wild-type AHA decrease in intensity or are even lost, whereas features typical of the dynamic signature of PPA can be detected (Figure 4 and 5). The only exception is AHA5 mutant in which more complex effects are induced on coupled motions, but not flexibility profiles, upon the introduction of the 5 mutations affecting weak intramolecular interactions. AHA mutants also present a modification of the salt- bridge interactions, their networks and distribution in clusters of spatial proximity (Figure 6 and 7). In particular, highly persistent salt bridges conserved in all the simulated variants can be detected (Figure 6 and 7; Table S1-group I in Supporting Information S1), which can be the necessary and sufficient elements, at least concerning ionic interactions, for the stabilization of the a-amylase fold. These results fit well in the context of recent MD studies demonstrating that, in the (b/a)8 barrel fold, coevolving residues with crucial role in function and fold stability are interconnected by intramolecular interactions, as well as they control the most important and conserved correlated and anti-correlated motions governing this fold [27]. Relevance of correlated motions in identifying clusters of critical residues for protein function and stability have also been shown in broader context and related to other protein folds [24,51,52]. In particular, in the group I of salt bridges (Table S1 in Supporting Information S1, Figure 6 and 7), it is relevant to mention the presence of a stable interaction between R338A and the residue E19A of the silent protease catalytic triad (387RP and E27P), the latter previously proposed as a relevant residue for AHA stability and belonging to the silent protease catalytic triad of Cl2 - dependent a-amylases [53]. Both AHA, mutants and PPA share a salt-bridge network (D174A-R172A-D84A and D197P-R195P-D96P) involving the catalytic nucleophile D174A (D197P), and relying on the hub role of R172A in mediating interactions both with D174A and D84A (Table S1 in Supporting Information S1, Figure 6 and 7). These networks could be another PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24214