%0 Journal Article
%T 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.
%A Iwaoka M
%A Kumakura F
%A Yoneda M
%A Nakahara T
%A Henmi K
%A Aonuma H
%A Nakatani H
%A Tomoda S
%J J Biochem
%V 144
%N 1
%D Jul 2008
%M 18407938
%F 3.241
%R 10.1093/jb/mvn049
%X Oxidative regeneration pathways of bovine pancreatic ribonuclease A (RNase A), which has four SS linkages, were studied at 25 degrees C and pH 8.0 by using trans-3,4-dihydroxy-1-selenolane oxide (DHS(ox)), a new selenoxide reagent with strong oxidation power. The short-term folding study using a quench-flow instrument ( approximately 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 ( approximately 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, DHS(ox) allowed facile identification of the conformational folding steps coupled with SS rearrangement on the major oxidative folding pathways.