Protein thermostability above 100°C: A key role for ionic interactions
- Costantino Vetriani*,
- Dennis L. Maeder*,
- Nicola Tolliday*,
- Kitty S.-P. Yip†,
- Timothy J. Stillman†,
- K. Linda Britton†,
- David W. Rice†,
- Horst H. Klump‡, and
- Frank T. Robb*,§
- *Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street, Baltimore, MD 21202; ‡Department of Biochemistry, University of Cape Town, Rondebosch, South Africa 7700; and †Krebs Institute, University of Sheffield, Sheffield 510 2TN, United Kingdom
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Edited by Max F. Perutz, Medical Research Council, Cambridge, United Kingdom, and approved August 10, 1998 (received for review April 22, 1998)
Abstract
The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100°C. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100°C and 88°C, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104°C. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104°C over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.
Footnotes
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↵ § To whom reprint requests should be addressed. e-mail: robb{at}umbi.umd.edu.
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This paper was submitted directly (Track II) to the Proceedings Office.
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Data deposition: The atomic coordinates for Thermococcus litoralis GluDH have been deposited in the Protein Data Bank, Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (PDB ID code 1BVU).
- ABBREVIATIONS:
- GluDH,
- glutamate dehydrogenase EC 1.4.1.3;
- T138E,
- Thr 138 replaced with Glu;
- D167T,
- Asp 167 replaced with Thr
- Copyright © 1998, The National Academy of Sciences
