Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase.
Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.
Jang
Do Soo
DS
National Research Laboratory of Protein Folding and Engineering, Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.
Lee
Hyeong Ju
HJ
Lee
Byeongdu
B
Hong
Bee Hak
BH
Cha
Hyung Jin
HJ
Yoon
Jinhwan
J
Lim
Kwanseop
K
Yoon
Ye Jeong
YJ
Kim
Jehan
J
Ree
Moonhor
M
Lee
Hee Cheon
HC
Choi
Kwan Yong
KY
D002942:Circular Dichroism; D019281:Dimerization; D011489:Protein Denaturation; D017510:Protein Folding; D016958:Pseudomonas putida / Q000201:enzymology*; D012542:Scattering, Radiation; D013251:Steroid Isomerases / Q000737:chemistry*; D014508:Urea / Q000737:chemistry
D014508:Urea; D013251:Steroid Isomerases