Nonlocal Interactions Are Responsible for Tertiary Structure Formation in Staphylococcal Nuclease

Abstract

Rapid molecular collapse mediated by nonlocal interactions is believed to be a crucial event for protein folding. To investigate the role of nonlocal interactions in tertiary structure formation, we performed a nonlocal interaction substitution mutation analysis on staphylococcal nuclease (SNase). Y54 and I139 of wild-type (WT) SNase and Delta140-149 were substituted by cysteine to form intramolecular disulfide bonds, respectively called WT-SS and Delta140-149-SS. Under physiological conditions, the reduced form of Delta140-149-SS appears to assume a denatured structure; in contrast, the oxidized form of Delta140-149-SS forms a native-like structure. From this result, we conclude that the C-terminal region participates in a nonlocal interaction that is indispensable for the native structure. Although the oxidized form of WT-SS assumes a more compact denatured structure under acidic conditions than the WT, the kinetic measurements reveal that the refolding reactions of both the reduced and oxidized forms of WT-SS are similar to those of the WT, suggesting that an intact nonlocal interaction is established within the dead time (22 ms). On the basis of these results, we propose that the native nonlocal contact established at the early stage of the folding process facilitates further secondary structure formation.

Cavities determine the pressure unfolding of proteins

“…Beyond such observations, by focusing on the pressure unfolding reaction proper, we have characterized, with unprecedented structural and energetic resolution, the pressure unfolding landscape of SNase and its variants. Deletion mutagenesis (33), H/D exchange (34,35), and mechanical unfolding experiments (36) all at atmospheric pressure have provided a consistent picture of the modular nature of SNase with two subdomains, an N-terminal β-barrel core domain that includes helix 1, and a C-terminal domain consisting primarily of helix 3, with helix 2 acting as an interface between the two subdomains and the C-terminal loop Leu-137 to Ser-141 locking the protein into its correct tertiary structure through long range contacts (37,38). Besides the typical cis/trans proline isomerization observed within the unfolded states on a slow time scale, multiphasic kinetics have been detected in a stopped-flow study of a proline-free variant of SNase (39), suggesting the accumulation of on-pathway intermediate states.…”

Fast Subdomain Folding Prior to the Global Refolding Transition of E. coli Adenylate Kinase: A Double Kinetics Study

“…These early folding events enforce constraints that drastically reduce the conformational space available to the ensemble of protein molecules, which in turn create bias toward the native structure. 6,35,36 This is a typical cross-linking effect.…”

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

“…100 and 300 μs). Kato et al (2010) studied the folding of mutants of S. nuclease, where NLIs were perturbed and concluded that the native NLIs established at the early stage of the folding process facilitate further secondary structure formation. Meisner and Sosnick (2004) studied the kinetics of folding of a two-chain engineered protein in which helicity and collision rate can be varied and concluded that an unstructured encounter complex can successfully initiate rapid folding, with helix formation occurring at a later stage.…”