HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: ps.04780704v1 13/12/3214    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tanaka, N. Articles by Kunugi, S. Search for Related Content PubMed PubMed Citation Articles by Tanaka, N. Articles by Kunugi, S. Social Bookmarking                   What's this?

Interaction of the N-terminal domain of Escherichia coli heat-shock protein ClpB and protein aggregates during chaperone activity

Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan

(RECEIVED March 31, 2004; FINAL REVISION July 22, 2004; ACCEPTED August 5, 2004)

The Escherichia coli heat-shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system. The ClpB N-terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown. In this study, we investigated the effect of the ClpB N-terminal domain on malate dehydrogenase (MDH) refolding. ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate. However, the refolding kinetics was not affected by deletion of the ClpB N-terminal domain (ClpBN), indicating that MDH refolding was affected by interaction with the N-terminal domain. In addition, the MDH refolding yield increased 50% in the presence of the ClpB N-terminal fragment (ClpBN). Fluorescence polarization analysis showed that this chaperone-like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH. The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 µM from the calculation of the refolding kinetics. Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone-like activity and the affinity to the substrate. In addition, these residues are involved in stimulation of ATPase activity in ClpB. Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP-induced ClpB conformational change.

Keywords: molecular chaperone; ClpB; chaperone-like activity; protein aggregate; refolding; amino acid substitution; substrate binding site

Abbreviations: ClpBN, N-terminal domain of ClpB • ClpBN, ClpB protein with a deleted N-terminal domain • MDH, malate dehydrogenase • GdnHCl, guanidine hydrochloride • K_d, dissociation constant

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04780704.

Reprint requests to: Naoki Tanaka, Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan; e-mail: tanaka{at}ipc.kit.ac.jp; fax: +81-75-724-7710.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				R. Lum, M. Niggemann, and J. R. Glover Peptide and Protein Binding in the Axial Channel of Hsp104: INSIGHTS INTO THE MECHANISM OF PROTEIN UNFOLDING J. Biol. Chem., October 31, 2008; 283(44): 30139 - 30150. [Abstract] [Full Text] [PDF]

				M. E. Barnett, M. Nagy, S. Kedzierska, and M. Zolkiewski The Amino-terminal Domain of ClpB Supports Binding to Strongly Aggregated Proteins J. Biol. Chem., October 14, 2005; 280(41): 34940 - 34945. [Abstract] [Full Text] [PDF]