Denatured State Loop Formation Thermodynamics of a Hybrid Polypeptide

Abstract

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...