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.

The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding kinetics, and residual structure under denaturing conditions have been investigated. The concentration dependence of thermal denaturation and size-exclusion chromatography indicate that UBA(1) is monomeric. Guanidine hydrochloride (GdnHCl) denaturation experiments reveal that the unfolding free energy, ΔG u°′(H2O), of UBA(1) is 2.4 kcal mol–1. Stopped-flow folding kinetics indicates sub-millisecond folding with only proline isomerization phases detectable at 25 °C. The full folding kinetics are observable at 4 °C, yielding a folding rate constant, k f, in the absence of a denaturant of 13,000 s–1 and a Tanford β-value of 0.80, consistent with a compact transition state. Evaluation of the secondary structure via circular dichroism shows that the residual helical structure in the denatured state is replaced by polyproline II structure as the GdnHCl concentration increases. Analysis of NMR secondary chemical shifts for backbone 15NH, 13CO, and 13Cα atoms between 4 and 7 M GdnHCl shows three islands of residual helical secondary structure that align in sequence with the three native-state helices. Extrapolation of the NMR data to 0 M GdnHCl demonstrates that helical structure would populate to 17–33% in the denatured state under folding conditions. Comparison with NMR data for a peptide corresponding to helix 1 indicates that this helix is stabilized by transient tertiary interactions in the denatured state of UBA(1). The high helical content in the denatured state, which is enhanced by transient tertiary interactions, suggests a diffusion–collision folding mechanism.

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.

Previous work with the four-helix bundle protein cytochrome c' from Rhodopseudomonas palustris using histidine-heme loop formation thermodynamic 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

Chamapsocephalus gunnari) have been investigated. A novel fluorescence assay was utilized that simultaneous monitors changes to the global protein structure, structural changes near the active site, and aggregation of the enzyme in response to increasing temperature and increasing concentration of the natural osmolyte, trimethyl amine N-oxide (TMAO), a stabilizer of protein structure. Using this assay, the reverse changes of stability and affinity for oxamate were established for both, phLDH and cgLDH. Importantly, a low-temperature (pre-denaturation) structural transition was found that precedes the high-temperature (denaturation) transition for both LDHs and coincides with increasing enzymatic activity. The structural transitions of the global protein structure and the active site are concerted for the rigid (phLDH) and not concerted for the flexible (cgLDH) LDHs. The profound contribution of entropy to G along with the higher structural flexibility increases functional plasticity of the psychrophilic cgLDH. TMAO increases stability and shifts all structural transitions to the higher temperatures for both orthologs and simultaneously reduces their catalytic activity. The multiple active and inactive along with intermediate substate conformations of the enzyme exist in equilibrium at the stage preceding irreversible thermal inactivation. This equilibrium is an essential selective factor for the adaptation of an enzyme to the environmental temperature. It seems also possible that thermal adaptation of proteins may be complemented by evolution of the cellular milieu. 285-Pos Board B50 Denatured State Loop Formation Thermodynamics of a HybridPolypeptide Previous work with the four-helix bundle protein cytochrome c' in Rhodopseudomonas palustris using histidine-heme loop formation thermodynamic methods revealed fold-specific deviations from random coil character in its denatured state ensemble. To examine the generality of this finding, we extend this work to a three-helix bundle polypeptide, the human DNA excision repair protein's second ubiquitin-associated (UBA) domain, UBA(2). We use yeast iso-1-cytochrome c as a scaffold, fusing the UBA(2) domain to the N-terminus of iso-1-cytochrome c. Using site-directed mutagenesis, we have engineered histidine into solvent accessible surface residue positions within the all-alpha fold, creating eight single histidine variants. Isothermal equilibration denaturation studies reveal that the fusion protein unfolds in a 3-state process, commencing with iso-1-cytochrome c followed by UBA(2). Thermodynamic stability experiments also demonstrate that the histidine residues in the UBA(2) domain strongly destabilize iso-1-cytochrome c. Furthermore, histidine-heme loop formation equilibria show lower apparent pK a 's compared to the pseudo-wild type variant, indicating significant interactions in the denatured state. We will compare the degree of deviation of loop stability versus loop size, relative to predictions of the Jacobson-Stockmayer relationship used in our previous work on c...