Denatured State Conformational Biases in Three-Helix Bundles Containing Divergent Sequences Localize near Turns and Helix Capping Residues

• Published in: Biochemistry (Easton) Vol. 60; no. 41; pp. 3071 - 3085
• Main Authors: Leavens, Moses J, Spang, Lisa E, Cherney, Melisa M, Bowler, Bruce E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.10.2021
• Subjects: Cytochromes c - chemistry; Cytochromes c - genetics; denaturation; Guanidine - chemistry; guanidines; histidine; humans; hydrophobicity; Kinetics; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Protein Denaturation - drug effects; Rhodopseudomonas palustris; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Thermodynamics; topology; yeasts
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.1c00400

Rhodopseudomonas palustris cytochrome c′, a four-helix bundle, and the second ubiquitin-associated domain, UBA(2), a three-helix bundle from the human homologue of yeast Rad23, HHR23A, deviate from random coil behavior under denaturing conditions in a fold-specific manner. The random coil deviations in each of these folds occur near interhelical turns and loops in their tertiary structures. Here, we examine an additional three-helix bundle with an identical fold to UBA(2), but a highly divergent sequence, the first ubiquitin-associated domain, UBA(1), of HHR23A. We use histidine–heme loop formation methods, employing eight single histidine variants, to probe for denatured state conformational bias of a UBA(1) domain fused to the N-terminus of iso-1-cytochrome c (iso-1-Cytc). Guanidine hydrochloride (GuHCl) denaturation shows that the iso-1-Cytc domain unfolds first, followed by the UBA(1) domain. Denatured state (4 and 6 M GuHCl) histidine–heme loop formation studies show that as the size of the histidine–heme loop increases, loop stability decreases, as expected for the Jacobson–Stockmayer relationship. However, loops formed with His35, His31, and His15, of UBA(1), are 0.6–1.1 kcal/mol more stable than expected from the Jacobson–Stockmayer relationship, confirming the importance of deviations of the denatured state from random coil behavior near interhelical turns of helical domains for facilitating folding to the correct topology. For UBA(1) and UBA(2), hydrophobic clusters on either side of the turns partially explain deviations from random coil behavior; however, helix capping also appears to be important.