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.
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.
Iwaoka
Michio
M
Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan. miwaoka@tokai.ac.jp
Kumakura
Fumio
F
Yoneda
Mitsumasa
M
Nakahara
Toshitaka
T
Henmi
Kayo
K
Aonuma
Hiromi
H
Nakatani
Hiroyasu
H
Tomoda
Shuji
S
D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D016566:Organoselenium Compounds / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012995:Solubility; D014867:Water / Q000737:chemistry
C531309:3,4-dihydroxy-1-selenolane oxide; D004220:Disulfides; D006573:Heterocyclic Compounds, 1-Ring; D007202:Indicators and Reagents; D016566:Organoselenium Compounds; D014867:Water; D012259:Ribonuclease, Pancreatic