Red to experimental data, predictions of pKa values within a couple of seconds. For the Apaf-1 and cytochrome c, PROPKA predicted the lysine residues to become Niaprazine Autophagy protonated (positively charged) whereas residues of aspartate and glutamate to become deprotonated (negatively charged). Certainly, this really is not often the case in proteins, and for buried, functionally relevant amino acid residues deviations from this rule had been described [96]. Having said that, so long as the residues that were implied within the formation of salt bridges between cytochrome c and Apaf-1 were exclusively surface situated, these trivial assumptions on their protonation states look to become reasonable. The pairs of neighboring acidic residues on the surface of Apaf-1 could, in principle, share a proton even in spite of their surface location. However, in the presence of a positively charged lysine residue (see Figs. two and three) even partial protonation of those carboxyl groups is incredibly unlikely for the reason that of simple electrostatic motives. Question 2. Referring to “dynamic nature” of interactions which can be observed in MD simulations, it could be exciting to analyze Fig. five in terms of significant states (long-living interactions) current in between corresponding residues. Authors’ response: We thank the reviewer for this comment. Indeed, the key feature in the interactions described is their dynamic nature; none of the contacts observed was long-living. Instead, every single distinct contact was lost after which regained at picoseconds. The only exceptions have been salt bridges between residues Lys25 and Asp941 too as Lys8 and Asp1147, which could be maintained for as much as ten ns, see Fig. 5. In the revised manuscript, we have updated Fig. five to contain the graph for distance in between Lys86 and Asp1064, and have rescaled the Y axis (distances) to superior illustrate the mobility of residues. To provide additional facts regarding the dynamic properties ofthe salt bridges, we’ve got added a brand new Table 3 in to the revised manuscript. Moreover, we plotted the distances involving proton donor and acceptor atoms of interacting residues against each other for every single in the 3 stable bifurcated bridges (see the new Fig. six). Query three. The binding of cytochrome C to WD domains on the apoptotic activating factor Apaf-1 is generalizedhypothesized within the discussion onto the possible function of WD domains in “transmitting mechanical signals as opposed to their purely structural role”. This idea should be explained and formulated in much more clear way. Authors’ response: We’ve got expanded the respective section of the Discussion.Reviewer’s report four: Prof. Gerrit Vriend, Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre, Nijmegen, The NetherlandsReviewer 4: I am not familiar with cytochrome c at all and poorly read-in on apoptosis, which, I guess, disqualifies me a little as a referee. But I will do my most effective. 1) As a bioinformatician, I normally get Disperse Red 1 Epigenetic Reader Domain worried when I read that protein structures got `improved’ by molecular dynamics. MD is really a good technique, but our YASARA experiences [85] made clear that MD generally drives structure models away in the true minimum. Authors’ response: We totally agree with the notion that MD simulations could possibly drive structures away from the correct power minima. As a result, in our article, we initial obtained energy minimized model structures and only then utilized MD simulations to tackle the dynamics of a number of them. In the revised version we’ve replaced `improved’ with a far more.
