Study of HubWA Protein Folding Reaction by Measuring the Stability of Folding Intermediate

[摘要] The contribution of hydrophobic residues to the protein folding reaction was studied by using HubWA variantproteins with I and L to V mutation. Folding kinetics of all V variant proteins was observed to be satisfied by a three-state onpathway mechanism, U ⇌ I ⇌ N, where U, I, and N represent unfolded, intermediate, and native state, respectively. Threestate folding reaction was quantitatively analyzed and the free energy of folding of each elementary reactions and overall folding reaction, ΔGoUI, ΔGoIN, and ΔGoUN, were obtained. From the ratio of free energy difference between the variant protein andHubWA, ΔΔGoUI/ΔΔGoUN (ΔΔGoUI = ΔGoUI (variant protein) − ΔGoUI (HubWA) and ΔΔGoUN = ΔGoUN (variant protein) − ΔGoUN(HubWA)), the contribution of hydrophobic residues to HubWA folding was analyzed. The residues which are located in thehydrophobic core between α-helix and β-sheet, I3, I13, L15, I30, L43, I61 and L67, showed ΔΔGoUI/ΔΔGoUN value of ~0.5when each of these residues was mutated to V, indicating that these residues form relatively solid hydrophobic core in theintermediate state. Residues located at the end of secondary structures and loop, I23, L69 and I36 showed ΔΔGoUI/ΔΔGoUNvalue below 0.4 when each of these residues was mutated to V, indicating that the region containing these residues are looselyformed in the intermediate state. V17A, L50V and L56V showed fairly high ΔΔGoUI/ΔΔGoUN value of ~0.8. Since L50 andL56 are located in the region containing long loop (residue 46 to 62), it is suggested that the high ΔΔGoUI/ΔΔGoUN value ofthese residues prevents the formation of aggregate at the early stage of folding reaction.

[学科分类] 化学（综合）

[关键词] Hydrophobic interactions and protein folding;Protein folding intermediate