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

Abstract

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.

High-Accuracy Prediction of Stabilizing Surface Mutations to the Three-Helix Bundle, UBA(1), with EmCAST

“…This difference may be attributed to the immediate availability of N-capping interactions by E176T, indicating that helix-capping interactions can promote efficient folding. Observations of helix-capping residues promoting structure in the denatured state further support this interpretation . In contrast, macroscopic dipole optimization by H192E will be available only after the formation of helix 3.…”

“…Observations of helix-capping residues promoting structure in the denatured state further support this interpretation. 27 In contrast, macroscopic dipole optimization by H192E will be available only after the formation of helix 3.…”

“…The domain is one of two UBA domains found in the human homologue of Saccharomyces cerevisiae Rad23, HHR23A, DNA excision repair protein . We have previously characterized the stability, folding kinetics, and denatured state properties of wild-type (WT) UBA(1) and determined its X-ray structure at a resolution of 1.60 Å. , The domain is of modest stability (2.4 kcal/mol), providing it as a good candidate for rational stabilization. Here, we experimentally characterize the structure, stability, and folding landscape of stabilized variants and compare the stabilizations of single and multisite variants to those predicted by EmCAST.…”

Residual Structure in the Denatured State of the Fast-Folding UBA(1) Domain from the Human DNA Excision Repair Protein HHR23A

“…ILV clusters involving branched, aliphatic side chains have been suggested to be cores of stability in partially unfolded states and may be a major contributor to the initiation of tertiary folding in UBA(1). In our recent study using His-heme loop formation to evaluate nonrandom structure in the denatured state of UBA(1), we showed that UBA(1) has a large ILV cluster capable of stabilizing the residual structure near both turns. The first turn of UBA(1) is one of the few sequence regions of UBA domains that is conserved across evolution .…”

DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science

“…We therefore studied the H/D-exchange behavior of unfolded human ubiquitin in 6 M GdmCl by the DMSO-quenched H/D-exchange 2D NMR method with the use of spin desalting columns [136]. Although the persistence of residual structures in unfolded proteins in concentrated denaturant has been reported for several different proteins [50,[147][148][149][150][151][152][153], the present method enabled us to estimate the p values of individually identified NH protons, including nonprotected NH protons in the N state [136].…”