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Journal of Biochemistry Advance Access published online on April 11, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn049
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© 2008 The Japanese Biochemical Society

Direct Observation of Conformational Folding Coupled with Disulfide Rearrangement by Using a Water-Soluble Selenoxide Reagent. A Case of Oxidative Regeneration of Ribonuclease A under Weakly Basic Conditions

Michio Iwaoka1,*, Fumio Kumakura1, Mitsumasa Yoneda1, Toshitaka Nakahara1, Kayo Henmi1, Hiromi Aonuma1, Hiroyasu Nakatani2 and Shuji Tomoda2

1 Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan;
2 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan

*To whom correspondence should be addressed. Tel: +81-463-58-1211, Fax: +81-463-50-2094, E-mail: miwaoka{at}tokai.ac.jp

Received March 8, 2008; Accepted April 8, 2008


  Abstract

Summary:

Oxidative regeneration pathways of RNase A, which has four SS linkages, were studied at 25 °C and pH 8.0 by using trans-3,4-dihydroxy-1-selenolane oxide (DHSox), a new selenoxide reagent with strong oxidation power. The short-term folding study using a quench-flow instrument (~1 min) revealed that early intermediates (1S, 2S, 3S, and 4S) are formed stochastically and irreversibly from the reduced protein (R) and do not have any stable structures. In the long-term folding study (~300 min), on the other hand, slow generation of the key intermediates (des[65–72] and des[40–95]) through SS rearrangement from the 3S intermediate ensemble was observed, followed by slight formation of native RNase A (N). The parallel UV and CD measurements demonstrated that formation of the key intermediates is accompanied with the formation of the native-like structures. Thus, DHSox allowed facile identification of the conformational folding steps coupled with SS rearrangement on the major oxidative folding pathways.

Key Words: CD, disulfide, protein folding, selenium, UV


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