Journal of Biochemistry Advance Access originally published online on April 11, 2008
Journal of Biochemistry 2008 144(1):121-130; doi:10.1093/jb/mvn049
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© 2008 The Japanese Biochemical Society
Direct Observation of Conformational Folding Coupled with Disulphide Rearrangement by Using a Water-soluble Selenoxide Reagent—A Case of Oxidative Regeneration of Ribonuclease A under Weakly Basic Conditions
1Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292; and 2Department 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
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Abstract |
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Oxidative regeneration pathways of bovine pancreatic ribonuclease A (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, disulphide, protein folding, selenium, UV
Abbreviations: 1S, 2S, 3S and 4S, ensembles of folding intermediates of RNase A with one, two, three and four SS linkages, respectively; AEMTS, 2-aminoethyl methanethiosulphonate; des[40–95], a folding intermediate of RNase A having three native SS linkages and lacking the Cys40–Cys95 SS linkage; des[65–72], a folding intermediate of RNase A having three native SS linkages and lacking the Cys65–Cys72 SS linkage; DHSox, DL-trans-3,4-dihydroxy-1-selenolane oxide; DHSred, DL-trans-3,4-dihydroxy-1-selenolane; DTTox, trans-4,5-dihydroxy-1,2-dithiane (oxidized dithiothreitol); DTTred, DL-dithiothreitol; N, the folded state of RNase A with four native SS linkages; R, a fully reduced state of RNase A; RNase A, bovine pancreatic ribonuclease A; SH, thiol; SS, disulphide