The change of conditions does not affect Ros87 downhill folding mechanism

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.

Structural characterization of the thermal unfolding pathway of human VEGFR1 D2 domain

“…Thermal unfolding studies may be very useful to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein [ 28 ]. Multinuclear NMR methodologies, coupled with DSC analysis, represent a powerful tool to describe protein thermal unfolding mechanisms at an atomic level [ 20 , 29 , 30 , 31 , 32 , 33 ]. The human VEGFR1D2 is an Ig domain of the I‐set containing one small β‐sheet involving strands B, E, and D, a second larger β‐sheet involving strands A', G, F, C, and C', and a short helical turn consisting of residues 199–201 in the crossover between strands E and F (Fig.…”

The Ros/MucR Zinc-Finger Protein Family in Bacteria: Structure and Functions

“…It was shown that the substitution of the native ion resulted in completely different folding scenarios. These results outline the complex cross-correlation between the protein–metal ion equilibrium and the folding mechanism, proposing this interplay as a key factor in the proper metal ion selection by a specific metallo-protein [ 25 ].…”

“…Furthermore, the prokaryotic ZF (as recently shown for Ros87 from A. tumefaciens ), which has a bigger βββαα domain with a larger hydrophobic core with respect to its eukaryotic counterpart, represents a valuable model protein to study metal ion interactions with metallo-proteins [ 25 ]. The recent data have demonstrated that the DNA-binding domain of A. tumefaciens Ros structurally tolerates the Ni(II) ion, albeit with important structural perturbations, but not Pb(II) and Hg(II), and this protein proved to be functional in vitro when the Zn(II) ion was replaced by Cd(II).…”

MucR from Sinorhizobium meliloti: New Insights into Its DNA Targets and Its Ability to Oligomerize

“…The structure and function of MucR and Ros have been extensively investigated. Specifically, the folding mechanism and metal coordination sphere of the prokaryotic zinc-finger present in Ros from Agrobacterium tumefaciens has been elucidated [6,[12][13][14][15][16][17]. In A. tumefaciens, Ros is involved in the Horizontal Gene Transfer (HGT) of genes from the bacteria to the host plant and acts as a repressor of virC and virD operons by binding to AT-rich sequences identified in the operon promoters [1,18,19].…”