A Localized Specific Interaction Alters the Unfolding Pathways of Structural Homologues

Abstract

Reductive unfolding studies of proteins are designed to provide information about intramolecular interactions that govern the formation (and stabilization) of the native state and about folding/ unfolding pathways. By mutating Tyr92 to G, A, or L in the model protein, bovine pancreatic ribonuclease A, and through analysis of temperature factors and molecular dynamics simulations of the crystal structures of these mutants, it is demonstrated that the markedly different reductive unfolding rates and pathways of ribonuclease A and its structural homologue onconase can be attributed to a single, localized, ring-stacking interaction between Tyr92 and Pro93 in the bovine variant. The fortuitous location of this specific stabilizing interaction in a disulfide-bond-containing loop region of ribonuclease A results in the localized modulation of protein dynamics which, in turn, enhances the susceptibility of the disulfide bond to reduction leading to an alteration in the reductive unfolding behavior of the homologues. These results have important implications for folding studies involving topological determinants to obtain folding/unfolding rates and pathways, for protein structure-function prediction through fold recognition, and for predicting proteolytic cleavage sites.

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

“…As expected, this value is significantly smaller than the Gibbs free energy of global protein unfolding which is reported to be in the range 18-40 kJ mol 21 (Refs. 3,4,32,33). On the other hand, the value of DG 0 derived for BSA is about 2.6 times lower than that reported for the two least reactive S-S bridges in BPTI 31 thus suggesting that the disulfide bridges in BSA are on average more exposed to the solvent.…”

Ribonucleases as potential modalities in anticancer therapy

“…The 30–75 disulfide bond in Onconase is more solvent exposed that other disulfide bonds in this enzyme or in RNase A (Narayan et al, 2004). Therefore, the reduction rate of this bond is10 3 -fold greater than that of the analogous 40–95 bond in RNase A (Xu et al, 2006). This disulfide bond substantially contributes to the enzyme stability since the des(30–75) folding intermediate (Gahl and Scheraga, 2009) and a recombinant variant, with Ala residues replacing Cys30 and Cys75 (Torrent et al, 2008) are less stable (T m values approximately 71°C).…”

New monoclinic form of bovine pancreatic ribonuclease A from a salt solution and comparison of intermolecular interactions in ribonucleases A

“…On the other hand, until now, only the T form in the space group P3 2 21 was reported from salt solutions except for the crystal of RNase A in the liganded form or a mutant [24,25].…”