Abstract
Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability. Downhill folders are estimated to be rather rare in nature as they miss an energetically substantial folding barrier that can protect against aggregation and proteolysis. We have previously demonstrated that the prokaryotic zinc finger protein Ros87 shows a bipartite folding/unfolding process in which a metal binding intermediate converts to the native structure through a delicate barrier-less downhill transition. Significant variation in folding scenarios can be detected within protein families with high sequence identity and very similar folds and for the same sequence by varying conditions. For this reason, we here show, by means of DSC, CD and NMR, that also in different pH and ionic strength conditions Ros87 retains its partly downhill folding scenario demonstrating that, at least in metallo-proteins, the downhill mechanism can be found under a much wider range of conditions and coupled to other different transitions. We also show that mutations of Ros87 zinc coordination sphere produces a different folding scenario demonstrating that the organization of the metal ion core is determinant in the folding process of this family of proteins.
Highlights
Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability
The Differential scanning calorimetry (DSC) thermogram shows a first broad, reversible endotherm centered at about 324 K followed by a second reversible broad endotherm centered at 360 K (Fig. 2C) pinpointing a two steps unfolding process separated by a detectable partially folded intermediate state
The body of data collected in the present work clearly shows that change of buffer, pH and ionic strength does not influence Ros87partly downhill unfolding mechanism
Summary
Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability. The study of different members of this protein family[24,25,26] allowed us to demonstrate that this protein architecture to functionally fold can substitute the structural ion with a different set of amino acids or can even surrogate the stabilizing role of the metal cofactor with a network of H-bonds and hydrophobic interactions[25,27,28,29] These differences among iso-structural proteins led to different mechanisms of folding: the metal lacking Ml452–151 folds/ unfolds with a classical two state mechanism while the coordination of the structural zinc confers to Ros[87] a Scientific Reports | (2020) 10:21067
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