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

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.

Scaling properties of glycine-rich sequences in guanidine hydrochloride solutions.

The intrinsic polymer properties of glycine-rich sequences are evaluated with a set of iso-1-cytochrome c variants with N-terminal inserts of the sequence (GGGGGK)(n) for n = 1-5. The thermodynamics and kinetics of His-heme loop formation are measured as a function of guanidine hydrochloride (GdnHCl) concentration for loop sizes ranging from 22 to 46 residues. The scaling exponent for loop formation, ν(3), evaluated using the Jacobson-Stockmayer equation is near 1.

Propensities of aromatic amino acids versus leucine and proline to induce residual structure in the denatured-state ensemble of iso-1-cytochrome c.

Histidine-heme loop formation in the denatured state of a protein is a sensitive means for probing residual structure under unfolding conditions. In this study, we use a host-guest approach to investigate the relative tendencies of different amino acids to promote residual structure under denaturing conditions. The host for this work is a 6-amino-acid insert of five alanines, followed by a lysine engineered immediately following a unique histidine near the N-terminus of yeast iso-1-cytochrome c.