particular, the flexibility of AHA mutants is not displaced toward the L3N part of the loop as in PPA (Figure 4), whereas more remarkable effects can be identified in the decreased flexibility and modification in cross-correlation patterns at the interface between L3 and L5 (observable for example in AHASS, AHAVF, AHANR and AHA5) (Figure 4 and 5). However, it is interesting to observe that the highest flexibility of AHA at the interface between L5 and L3 is strongly reduced in the mutant variants with some of them displacing the flexibility toward more exposed regions of the L5 loop. In particular, the major modifications in the dynamic properties of AHA mutants, at this site, rely on differences in the salt bridge network mediated by D203A (E207A-K177A-D203A- K224A-E222A) (Figure 7), which is interrupted in some mutants and in PPA, (lacking D203A-K177A) as well as on the appearance in the AHA mutants of a salt bridge between residues E213A and R133A (whereas in PPA the corresponding glutamate residue, E249P, provide a salt bridge in the same area but with different orientation mediated by R158P), along with a general weakening of the cross-correlation networks in this region (Figure 8C). In fact, most of the AHA mutants lack or attenuate the correlated motions (Figure 8C) connecting residues in the 128–130A region with residues 204–206A. Moreover, some AHA mutants lose correla- tion toward E222A (belonging to the salt bridge cluster mediated by D203A). In the same area, PPA conserves only correlated motions involving K200P (the homologous residue to K177A). Moreover, AHA and its mutants maintained in this regions a salt bridge cluster between D126A-R131A-D130A which is absent in PPA, due to deletion and amino acidic substitutions. In summary, the most relevant differences between AHA and PPA in 128–130A and 204–206A regions are Q204A (L237P), G128A (N152P) D126A (S150P) and D130A, R131A (missing in PPA). Moreover, in PPA, the displacement of flexibility toward the N-terminal portion of L3 loop is also related to the presence of two salt bridge clusters which are absent, and not allowed to be restored due to deletion or substitutions, in AHA and its mutants (D159P-K140P-E171P and D138P-R214P). K106A is the corre- sponding residue of R214P but mediates in AHA and its mutants interactions with E138A (Q161P). In order to re-establish a PPA-like behavior in AHA mutants, a more complex network of interactions and communicating residues should be restored in this area, including pivotal residues in L3 which allow the flexibility to be displaced at the N-terminal extremity of the loop. Otherwise, mutations of AHA residues at the interface between L3 and L5 could be included to more successfully abolish the network of cross-correlated motions between the two loops, which is lacking in PPA. Domain C: a structural domain shielding hydrophobic residues of the catalytic domain Domain C is composed of 8 b strands arranged in a greek-key motif and tightly packed against a6, a7 and a 8 of the barrel (Figure 1). The interface between domains C and A is rich in aromatic and hydrophobic residues which contribute to the stabilization of the interdomain interface, as well as they mediate the extension of the hydrophobic core of domain A towards domain C [33]. The sandwich-like structure of domain C features, in both AHA and PPA, asymmetry between the two composing b- sheets: the buried b-sheet constitutes the interface with domain A and is stable and ordered, while the solvent-exposed b-sheet is highly disordered, and split into 2 separate 2-stranded b-sheets (Figure S9 in Supporting Information S1). The latter ones are highly persistent in PPA, whereas in AHA main chain H-bonds stabilizing the central part of these sheets are weakened. Interestingly, most of the mutants display PPA-like H-bond Molecular Dynamics of Psychrophilic Enzyme Mutants pattern (with the exception of AHA5SS) and the presence of a PPA-like salt bridge involving the C-terminal residues of the proteins, along with disappearance of some salt bridges at the interface between domain A and C typical of AHA (Figure 7; Table S1 in Supporting Information S1). This behaviour was unexpected. In fact, domain C does not carry any of the studied mutations, and nevertheless, shows structural and dynamical differences in mutated forms, demonstrating that single mutations have global and long range effects, affecting regions very distant from the site of mutation. Discussion Intramolecular weak interactions have a fundamental role in stabilizing protein structures. Special attention has been given to their role in the context of thermal adaptation of proteins [7,44,60], with particular regard to electrostatic interactions [46,61–64]. The present study provides molecular details related to the observed variations in thermal stability and kinetic parameters of AHA mutants with respect to the wild-type cold- and warm-adapted counterparts, as well as it points out long range effects induced by the mutations. Our MD investigation shows that the AHA mutations are capable of eliciting effects, in agreement with the restoring or strengthening of the target interactions, on the dynamic environ- ment of the mutated residues in a mesophilic-like direction. If the minor entity of the introduced mutations is considered, since few residues in a multi-domain protein of about 500 amino acids are mutated, it is striking to observe the wide range of different dynamical behaviours these mutants exhibit. Interestingly, the local effects, in the mutation sites, are also able to modify the dynamic character of the mutants, producing complex distal effects that underlie the intimate interplay between hydrophobicity, electrostatic interactions and protein dynamics. The mutations are extremely effective in modifying AHA structural and flexibility properties, as hypothesized by the experimental characterization [37], showing also that, in the case of multiple mutants, the modification of activity and thermal stability to mesophilic values is strongly advantaged. The ability of AHA mutations to elicit a mesophilic-like behaviour is confirmed in the results of MD simulations, where the comparison is restricted exclusively to structural and dynamic properties. In fact, the mutants exhibit secondary structure persistence, flexibility, correlated motions, electrostatic interactions, and, hydrophobicity in-between of those seen for the two wild-type enzymes. However, fundamental differences both in the structure and the sequence of the two wild-type enzymes still exist and make it impossible for these mutations alone to reproduce the full set of stabilizing local interactions seen in PPA. In fact, our investigation also shown, in excellent agreement with the experimental values, that the conversion of AHA in a mesophilic enzyme cannot be completely achieved by the introduction of these mutations alone. In fact, other substitutions, highlighted in our study, should be considered to get more efficient modification of AHA toward the mesophilic counterpart, as summarized in Figure 8D, as K334A or I367P. Our MD simulations of AHA mutant variants and their comparison to dynamic features of the wild-type counterparts also point out other long range effects induced by the mutations. In particular, the effect of flexibility is largely apparent in regions featuring high flexibility in AHA (loop 7) and PPA (loop 8), which mainly cluster in regions near the active site and substrate-binding groove, and long range effects are even transmitted to the C- terminal domain. In this context, the present investigation PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24214