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J. Biol. Chem., Vol. 283, Issue 14, 9300-9307, April 4, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/14/9300    most recent M710057200v1 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 Google Scholar Articles by Okuda, H. Articles by Matsuda, K. PubMed PubMed Citation Articles by Okuda, H. Articles by Matsuda, K. Social Bookmarking                      What's this?

The Intermediate Domain Defines Broad Nucleotide Selectivity for Protein Folding in Chlamydophila GroEL1^*

Hiroshi Okuda, Chihaya Sakuhana, Risa Yamamoto, Yuko Mizukami, Rika Kawai, Yusuke Sumita, Motoki Koga, Mutsunori Shirai, and Kazuhiko Matsuda¹

From the Department of Applied Biological Chemistry, School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan and the Department of Microbiology, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi 755-8505, Japan

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln⁴⁰⁰ and Ile⁴⁰⁴ in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.

Received for publication, December 10, 2007 , and in revised form, January 28, 2008.

^* This work was supported in part by the Program for Basic Research Activities for Innovative Biosciences (Bio-oriented Technology Research Advancement Institution) of Japan and by the "Academic Frontier" Project for Private Universities Matching Fund Subsidy from the Ministry of Education, Culture, Sports, Science, and Technology (2004-2008). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedures", Figs. S1-S3, Tables S1-S3, and Refs. 1-3.

¹ To whom correspondence should be addressed. Tel.: 81-742-43-1511 (ext. 3306); Fax: 81-742-43-1445; E-mail: kmatsuda{at}nara.kindai.ac.jp.

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