Reviewer 2's Review

“The paper by Rodrígez-Almazán et al raises an interesting question, how nonspecific protein-protein interactions influence the folding and stability of proteins. The interactions of various TIMs were chosen as a model system for the study, which is of general interest. The authors claim that macromolecular crowding influence the properties of the individual proteins not only due to the effect of the excluded volume of the cells but by helping of nonspecific protein-protein interactions. The idea is logic; the question is how the experimental data support it.
One of the methods applied by the authors is the study of the reactivation of the enzymes after denaturation. They are experts on this field and the experiments were carefully done. The reviewer accept that these results prove the stabilizing and protecting effect of the various functionally unrelated proteins on the TIM enzymes from different sources, however, he is not convinced that these effects are "nonspecific". Of course, they are not "specific" as, e.g., an enzyme - substrate interaction but all the proteins applied stabilize specifically the dimeric form of TIMs. One can imagine that e.g. lysozyme could interact preferentially with the inactive monomeric forms of the enzymes, shifting the equilibria towards them. The authors themselves cite a literature data concerning it (Ref. 9.) I accept that this stabilizing phenomenon can work in vivo, where the high apparent concentration of the proteins (cf. crowding) favor the association of the oligomers, which can be further stabilized by nonspecific interactions with other proteins.

However, the experimental system used in the present study was a relatively diluted system. Thus I guess that the last sentence of the Abstract " the data are suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins" is true but in the Discussion session the authors should be more cautious in the interpretation of their data.

In the second type of the experiments the authors try to provide direct evidence of the physical interaction between TIM and one of the interacting partners, lysozyme. It is not an easy task, taking into account the weakness of the interaction and the low protein concentrations. This is the reason that the authors used several order of magnitude higher concentrations in these experiments. I am afraid that the ITC is not the best method to demonstrate the interaction but I accept that it is hard to find a better one. It would be interesting if the authors could estimate the strength of the interaction.

My feeling is that considering the very low binding affinity and the low protein concentrations in the renaturation and stability experiments, no complex formation can be expected in those cases. I suggest discussing this problem.”

N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.