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Characterization of Kinetic Folding Intermediates of Recombinant Canine Milk Lysozyme by Stopped-Flow Circular Dichroism

Nakao, Masaharu and Maki, Kosuke and Arai, Munehito and Koshiba, Takumi and Nitta, Katsutoshi and Kuwajima, Kunihiro (2005) Characterization of Kinetic Folding Intermediates of Recombinant Canine Milk Lysozyme by Stopped-Flow Circular Dichroism. Biochemistry, 44 (17). pp. 6685-6692. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20170131-095829577

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Abstract

The intermediate in the equilibrium unfolding of canine milk lysozyme induced by a denaturant is known to be very stable with characteristics of the molten globule state. Furthermore, there are at least two kinetic intermediates during refolding of this protein:  a burst-phase (first) intermediate formed within the dead time of stopped-flow measurements and a second intermediate that accumulates with a rate constant of 22 s-1. To clarify the relationships of these intermediates with the equilibrium intermediate, and also to characterize the structural changes of the protein during refolding, here we studied the kinetic refolding reactions using stopped-flow circular dichroism at 10 different wavelengths and obtained the circular dichroism spectra of the intermediates. Comparison of the circular dichroism spectra of the intermediates, as well as the absence of observed kinetics in the refolding from the fully unfolded state to the equilibrium intermediate, has demonstrated that the burst-phase intermediate is equivalent to the equilibrium intermediate. The difference circular dichroism spectrum that represented changes from the kinetic intermediate to the native state had characteristics of an exciton coupling band, indicating that specific packing of tryptophan residues in this protein occurred in this phase. From these findings, we propose a schematic model of the refolding of canine milk lysozyme that is consistent with the hierarchical mechanism of protein folding.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi050082+DOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi050082%2BPublisherArticle
Additional Information:© 2005 American Chemical Society. Received 14 January 2005. Published online 7 April 2005. Published in print 1 May 2005. This work was supported by Grants-in-Aid for Scientific Research in Priority Areas (15076201) from the Ministry of Education, Culture, Science and Technology of Japan. We thank Mr. Takayuki Morimoto for his help in the preparation of canine milk lysozyme.
Funders:
Funding AgencyGrant Number
Japan Society for the Promotion of Science (JSPS)15076201
Ministry of Education, Culture, Sports, Science and Technology (MEXT)UNSPECIFIED
Issue or Number:17
Record Number:CaltechAUTHORS:20170131-095829577
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170131-095829577
Official Citation:Characterization of Kinetic Folding Intermediates of Recombinant Canine Milk Lysozyme by Stopped-Flow Circular Dichroism Masaharu Nakao, Kosuke Maki, Munehito Arai, Takumi Koshiba, Katsutoshi Nitta, and Kunihiro Kuwajima Biochemistry 2005 44 (17), 6685-6692 DOI: 10.1021/bi050082+
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73866
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:31 Jan 2017 18:44
Last Modified:03 Oct 2019 16:32

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