論文

査読有り
1997年7月

Temperature control for kinetic refolding of heat-denatured ovalbumin

PROTEIN SCIENCE
  • F Tani
  • ,
  • N Shirai
  • ,
  • T Onishi
  • ,
  • F Venelle
  • ,
  • K Yasumoto
  • ,
  • E Doi

6
7
開始ページ
1491
終了ページ
1502
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1002/pro.5560060713
出版者・発行元
CAMBRIDGE UNIV PRESS

The folding of heat-denatured ovalbumin, a non-inhibitory serpin with a molecular size of 45 kDa, was examined. Ovalbumin was heat-denatured at 80 degrees C under nonreducing conditions at pH 7.5 and then cooled either slowly or rapidly. Slow cooling allowed the heat-denatured ovalbumin to refold to its native structure with subsequent resistance to digestion by trypsin. Upon rapid cooling, by contrast, the heat-denatured molecules assumed the metastable non-native conformations that were susceptible to trypsin. The non-native species were marginally stable for several days at a low temperature, but the molecules were transformed slowly into the native conformation. Considering data from size-exclusion chromatography and from analyses of CD, intrinsic tryptophan fluorescence, and adsorption of the dye 1-anilinonaphthalene-8-sulfonate, we postulated that the non-native species that accumulated upon rapid cooling were compact but structureless globules with disordered side chains collectively as a folding intermediate. Temperature-jumped CD experiments revealed biphasic kinetics for the refolding process of heat-denatured ovalbumin, with the features of increasing and subsequently decreasing amplitude of the rapid and the slow phases, respectively, with the decrease in folding temperature. The temperature dependence of the refolding kinetics indicated that the yield of renaturation was maximal at about 55 degrees C. These findings suggested the kinetic partitioning of heat-denatured ovalbumin between alternative fates, slow renaturation to the native state and rapid collapse to the metastable intermediate state. Analysis of disulfide pairing revealed the formation of a scrambled form with non-native disulfide interactions in both the heat-denatured state and the intermediate state that accumulated upon rapid cooling, suggesting that non-native disulfide pairing is responsible for the kinetic barriers that retard the correct folding of ovalbumin.

リンク情報
DOI
https://doi.org/10.1002/pro.5560060713
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/9232650
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:A1997XK77600013&DestApp=WOS_CPL
ID情報
  • DOI : 10.1002/pro.5560060713
  • ISSN : 0961-8368
  • PubMed ID : 9232650
  • Web of Science ID : WOS:A1997XK77600013

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