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Temperature control for kinetic refolding of heat-denatured ovalbumin.
Protein Sci. 1997 Jul; 6(7):1491-502.PS

Abstract

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.

Authors+Show Affiliations

Research Institute for Food Science, Kyoto University, Japan. tani@soya.food.kyoto-u.ac.jpNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9232650

Citation

Tani, F, et al. "Temperature Control for Kinetic Refolding of Heat-denatured Ovalbumin." Protein Science : a Publication of the Protein Society, vol. 6, no. 7, 1997, pp. 1491-502.
Tani F, Shirai N, Onishi T, et al. Temperature control for kinetic refolding of heat-denatured ovalbumin. Protein Sci. 1997;6(7):1491-502.
Tani, F., Shirai, N., Onishi, T., Venelle, F., Yasumoto, K., & Doi, E. (1997). Temperature control for kinetic refolding of heat-denatured ovalbumin. Protein Science : a Publication of the Protein Society, 6(7), 1491-502.
Tani F, et al. Temperature Control for Kinetic Refolding of Heat-denatured Ovalbumin. Protein Sci. 1997;6(7):1491-502. PubMed PMID: 9232650.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Temperature control for kinetic refolding of heat-denatured ovalbumin. AU - Tani,F, AU - Shirai,N, AU - Onishi,T, AU - Venelle,F, AU - Yasumoto,K, AU - Doi,E, PY - 1997/7/1/pubmed PY - 1997/7/1/medline PY - 1997/7/1/entrez SP - 1491 EP - 502 JF - Protein science : a publication of the Protein Society JO - Protein Sci VL - 6 IS - 7 N2 - 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. SN - 0961-8368 UR - https://www.unboundmedicine.com/medline/citation/9232650/Temperature_control_for_kinetic_refolding_of_heat_denatured_ovalbumin_ L2 - https://doi.org/10.1002/pro.5560060713 DB - PRIME DP - Unbound Medicine ER -