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) 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 Google Scholar Google Scholar Articles by Frankenberg, R. J. Articles by Clark, D. S. Search for Related Content PubMed PubMed Citation Articles by Frankenberg, R. J. Articles by Clark, D. S. Social Bookmarking                   What's this?

Chemical denaturation and elevated folding temperatures are required for wild-type activity and stability of recombinant Methanococcus jannaschii 20S proteasome

¹ Joint Graduate Group in Bioengineering, University of California, San Francisco/University of California, Berkeley, San Francisco, California 94143, USA
² Department of Chemical Engineering, University of California, Berkeley, California 94720, USA
³ Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

Reprint requests to: Douglas S. Clark, Department of Chemical Engineering, 201 Gilman Hall, University of California, Berkeley, CA 94720, USA; e-mail: clark{at}cchem.berkeley.edu;fax: 510-643-1228.

The 20S proteasome from the extreme thermophile Methanococcus jannaschii (Mj) was purified and sequenced to facilitate production of the recombinant proteasome in E. coli. The recombinant proteasome remained in solution at a purity level of 80–85% (according to SDS PAGE) following incubation of cell lysates at 70°C. Temperature–activity profiles indicated that the temperature optima of the wild-type and recombinant enzymes differed substantially, with optimal activities occurring at 119°C and 95°C, respectively. To ameliorate this discrepancy, two recombinant enzyme preparations were produced, each of which included denaturation of the proteasome by 4 M urea followed by high-temperature (85°C) dialysis. The wild-type temperature optimum was restored, but only if proteasome subunits were denatured and refolded prior to assembly (a preparation designated as & ß). In contrast, when proteasome assembly preceded denaturation (designated + ß) the optimum temperature was raised to a lesser degree. Moreover, the & ß and + ß preparations had apparent thermal half-lives at 114°C of 54.2 and 26.2 min, respectively, and the thermostability of the less stable enzyme was more sensitive to a reduction in pH. Attainment of wild-type activity and stability thus required the proper folding of both the - and ß-subunits prior to proteasome assembly. Consistent with this behavior, dual-scanning calorimetry (DSC) measurements revealed differences in the reassembly efficiency of the two proteasome preparations. The ability to produce structural conformers with dramatically different thermal optima and thermostabilities may facilitate the determination of molecular forces and structural motifs responsible for enzyme thermostablity and high-temperature activity.

Keywords: Extremophilic recombinant enzymes; protein folding; thermostability

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