Probing Denatured State Conformational Bias in a Three-Helix Bundle, UBA(2), Using a Cytochrome c Fusion Protein

Moses J. Leavens, Melisa M. Cherney, Michaela L. Finnegan, Bruce E. Bowler

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Previous work with the four-helix-bundle protein cytochrome c′ from Rhodopseudomonas palustris using histidine-heme loop formation methods revealed fold-specific deviations from random coil behavior in its denatured state ensemble. To examine the generality of this finding, we extend this work to a three-helix-bundle polypeptide, the second ubiquitin-associated domain, UBA(2), of the human DNA excision repair protein. We use yeast iso-1-cytochrome c as a scaffold, fusing the UBA(2) domain at the N-terminus of iso-1-cytochrome c. We have engineered histidine into highly solvent accessible positions of UBA(2), creating six single histidine variants. Guanidine hydrochloride denaturation studies show that the UBA(2)-cytochrome c fusion protein unfolds in a three-state process with iso-1-cytochrome c unfolding first. Furthermore, engineered histidine residues in UBA(2) strongly destabilize the iso-1-cytochrome c domain. Equilibrium and kinetic histidine-heme loop formation measurements in the denatured state at 4 and 6 M guanidine hydrochloride show that loop stability decreases as the size of the histidine-heme loop increases, in accord with the Jacobson-Stockmayer equation. However, we observe that the His27-heme loop is both more stable than expected from the Jacobson-Stockmayer relationship and breaks more slowly than expected. These results show that the sequence near His27, which is in the reverse turn between helices 2 and 3 of UBA(2), is prone to persistent interactions in the denatured state. Therefore, consistent with our results for cytochrome c′, this reverse turn sequence may help to establish the topology of this fold by biasing the conformational distribution of the denatured state.

Original languageEnglish
Pages (from-to)1711-1721
Number of pages11
Issue number11
StatePublished - Mar 20 2018


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