Red to experimental information, predictions of pKa values inside a couple of seconds. For the Apaf-1 and cytochrome c, PROPKA predicted the lysine residues to be protonated (positively charged) whereas residues of aspartate and glutamate to become deprotonated (negatively charged). Needless to say, this can be not constantly the case in proteins, and for buried, functionally relevant amino acid residues deviations from this rule were described [96]. Having said that, provided that the residues that were implied inside the formation of salt bridges involving cytochrome c and Apaf-1 were exclusively surface located, these trivial assumptions on their protonation states look to be affordable. The pairs of neighboring acidic residues around the surface of Apaf-1 could, in principle, share a proton even in spite of their surface location. Nevertheless, within the KI-7 Data Sheet presence of a positively charged lysine residue (see Figs. 2 and 3) even partial protonation of these carboxyl groups is incredibly unlikely for the reason that of straightforward electrostatic motives. Question two. Referring to “dynamic nature” of interactions that may be observed in MD simulations, it will be fascinating to analyze Fig. 5 in terms of main states (long-living interactions) existing among corresponding residues. Authors’ response: We thank the reviewer for this comment. Certainly, the key feature in the interactions described is their dynamic nature; none on the contacts observed was long-living. Alternatively, every single certain get in touch with was lost and then regained at picoseconds. The only exceptions were salt bridges amongst residues Lys25 and Asp941 at the same time as Lys8 and Asp1147, which could possibly be maintained for up to 10 ns, see Fig. 5. In the revised manuscript, we have updated Fig. five to involve the graph for distance between Lys86 and Asp1064, and have rescaled the Y axis (distances) to far better illustrate the mobility of residues. To supply additional info about the dynamic properties ofthe salt bridges, we have added a brand new Table 3 in to the revised manuscript. Furthermore, we plotted the distances among proton donor and acceptor atoms of interacting residues against each other for every single in the three stable bifurcated bridges (see the new Fig. 6). Question 3. The binding of cytochrome C to WD domains of the apoptotic activating issue Apaf-1 is generalizedhypothesized inside the discussion onto the possible function of WD domains in “transmitting mechanical signals in lieu of their purely structural role”. This concept really should be explained and formulated in a lot more clear way. Authors’ response: We’ve got expanded the respective section of the Discussion.Reviewer’s report 4: Prof. Gerrit Vriend, Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre, Nijmegen, The NetherlandsReviewer 4: I’m not familiar with cytochrome c at all and poorly read-in on apoptosis, which, I guess, disqualifies me a bit as a referee. But I’ll do my greatest. 1) As a bioinformatician, I normally get worried when I study that protein structures got `improved’ by molecular dynamics. MD can be a good technique, but our YASARA experiences [85] made clear that MD generally drives structure models away in the correct minimum. Authors’ response: We fully agree together with the notion that MD simulations may possibly drive structures away in the accurate power minima. Hence, in our article, we initially obtained energy minimized model structures and only then employed MD simulations to tackle the dynamics of a few of them. Within the revised version we’ve Flufiprole References replaced `improved’ with a more.