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Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase
Author(s) -
Jang Do Soo,
Lee Hyeong Ju,
Lee Byeongdu,
Hong Bee Hak,
Cha Hyung Jin,
Yoon Jinhwan,
Lim Kwanseop,
Yoon Ye Jeong,
Kim Jehan,
Ree Moonhor,
Lee Hee Cheon,
Choi Kwan Yong
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2006.06.069
Subject(s) - small angle x ray scattering , chemistry , circular dichroism , sedimentation equilibrium , crystallography , ketosteroid , ultracentrifuge , equilibrium unfolding , sedimentation coefficient , urea , nuclear magnetic resonance spectroscopy , spectroscopy , monomer , scattering , isomerase , stereochemistry , chromatography , polymer , organic chemistry , enzyme , physics , quantum mechanics , optics
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 18.7 Å in 0 M urea, 17.3 Å in 5.2 M urea, and 25.1 Å in 7 M urea by SAXS. The size of KSI in 5.2 M urea was significantly decreased compared with those in 0 M and 7 M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2 M 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.