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Journal of Biochemistry Advance Access published online on April 27, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn058
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© 2008 The Japanese Biochemical Society

NMR studies on the equilibrium unfolding of ketosteroid isomerase by urea

Hyeong Ju Lee1,a, Do Soo Jang2,a,{dagger}, Hyung Jin Cha2, Hye Seon Moon1, Bee Hak Hong2, Kwan Yong Choi2,* and Hee Cheon Lee1,*

1Department of Chemistry, 2Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea, 790-784

*Author to whom correspondence should be addressed: Prof. Hee Cheon Lee, Department of Chemistry, Pohang University of Science and Technology, Pohang, Korea 790-784, E-mail: hcl{at}postech.ac.kr, Fax: 82-54-279-3399. Kwan Yong Choi, Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, Korea 790-784, E-mail: kchoi{at}postech.ac.kr, Fax: 82-54-279-2199

Received February 26, 2008; Accepted April 17, 2008


  Abstract

Summary

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of 1H-15N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the β5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the β6-strand. The results thus suggest that the interface region primarily around the β5- and β6-strands could play an important role as the starting positions in the unfolding process of KSI.

Key Words: Dimeric protein, Equilibrium unfolding, Ketosteroid isomerase, NMR, Urea

aThese two authors are equally contributed to this work.

{dagger}Present address: The J. David Gladstone Institute, SF, CA94158, USA


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