중간단계의 구조적 안정성을 통한 HubWA 단백질의 접힘(folding) 반응 탐색

The contribution of hydrophobic residues to the protein folding reaction was studied by using HubWA variant proteins with I and L to V mutation. Folding kinetics of all V variant proteins was observed to be satisfied by a three-state on-pathway mechanism, U ⇌ I ⇌ N, where U, I, and N represent unfolded, intermediate, and native state, respectively. Three-state 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 and HubWA, 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 the hydrophobic core between α-helix and β-sheet, I3, I13, L15, I30, L43, I61 and L67, showed ΔΔGoUI/ΔΔGoUN value of ~0.5 when each of these residues was mutated to V, indicating that these residues form relatively solid hydrophobic core in the intermediate state. Residues located at the end of secondary structures and loop, I23, L69 and I36 showed ΔΔGoUI/ΔΔGoUN value below 0.4 when each of these residues was mutated to V, indicating that the region containing these residues are loosely formed in the intermediate state. V17A, L50V and L56V showed fairly high ΔΔGoUI/ΔΔGoUN value of ~0.8. Since L50 and L56 are located in the region containing long loop (residue 46 to 62), it is suggested that the high ΔΔGoUI/ΔΔGoUN value of these residues prevents the formation of aggregate at the early stage of folding reaction. HubWA 단백질을 모델로 삼아 소수성 아미노산이 folding 반응에 끼치는 영향을 탐색하기 위하여 HubWA에 있는 I와 L을 V로 치환한 변이 단백질의 folding kinetics를 측정하였다. 변이 단백질의 folding kinetics는 HubWA 단백질과 마찬가지로 three-state on-pathway mechanism(U ⇌ I ⇌ N, U는 unfolded 상태, I는 중간단계, N은 native 상태를 의미한다)을 따르는 것으로 나타났다. Folding kinetics 분석을 통하여 three-state 반응의 elementary 반응과 전체 반응의 자유에너지인 ΔGoUI, ΔGoIN, ΔGoUN을 얻었고, 변이 단백질의 자유에너지와 HubWA 단백질의 자유에너지의 차(ΔΔGoUI = ΔGoUI(변이 단백질) - ΔGoUI(HubWA), ΔΔGoUN = ΔGoUN(변이 단백질) - ΔGoUN(HubWA))의 비인 ΔΔGoUI/ΔΔGoUN를 통하여 중간단계가 전체 folding 반응에 끼치는 영향을 각 소수성 잔기 별로 알아볼 수 있었다. HubWA의 입체구조에서 α-helix와 β-sheet가 상호작용하는 소수성 코어에 위치하는 아미노산인 I3, I13, L15, I30, L43, I61, L67을 V로 치환한 변이 단백질의 ΔΔGoUI/ΔΔGoUN 값이 ~0.5로 나타난 점은 이들 아미노산이 중간단계에서 native 상태보다는 느슨하지만 비교적 견고한 구조를 이루는 것으로 해석되었다. HubWA 입체구조에서 α-helix의 아미노말단에 위치하는 I23, 특정 이차구조가 없는 부위에 위치하는 I36, β-strand 5의 카복실말단에 위치하는 L69