Skip Navigation



PEDS Advance Access published online on March 29, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh027
This Article
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
17/3/285    most recent
gzh027v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Simler, B. R.
Right arrow Articles by Matthews, C. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Simler, B. R.
Right arrow Articles by Matthews, C. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Received November 25, 2003
Accepted March 4, 2004
Oxford University Press 1741-0134

Article

Zinc Binding Drives the Folding and Association of the Homo-trimeric {gamma}-Carbonic Anhydrase from Methanosarcina thermophila

B. Robert Simler 1, Brandon L. Doyle 2, and C. Robert Matthews 1*

1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605
2 Department of Chemistry and Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park, PA 16802

* To whom correspondence should be addressed. E-mail: c.robert.matthews{at}umassmed.edu.


  Abstract

Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homotrimeric enzyme, whose left-handed b-helical subunits bind three catalytic Zn2+ ions at symmetry-related subunit interfaces. The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of ~70 kcal (mol·trimer)-1 at standard state. Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well-described by a three-state model involving the folded apo-trimer, the folded monomer, and the unfolded monomer. The monomer is estimated to have a stability of 4.0 ± 0.3 kcal (mol·monomer)-1. The association to form apo-trimer contributes 13.2 ± 0.4 kcal (mol·trimer)-1, a value confirmed by analytical ultracentrifugation measurements. Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer. Literature values for the free energy of binding of one Zn2+ ion to a canonical active site, 16.4 kcal mol-1 (DiTusa et al., 2001), are consistent with the presumption that the >45 kcal (mol·trimer)-1 generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam.

Keywords: {beta} helix/carbonic anhydrase/folding and assembly of trimeric proteins/zinc binding


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Bacteriol.Home page
S. S.-W. Cot, A. K.-C. So, and G. S. Espie
A Multiprotein Bicarbonate Dehydration Complex Essential to Carboxysome Function in Cyanobacteria
J. Bacteriol., February 1, 2008; 190(3): 936 - 945.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.