Skip Navigation

This Article
Right arrow FREE Full Text (PDF) Freely available
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 Dabrowski, M. J.
Right arrow Articles by Atkins, W. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dabrowski, M. J.
Right arrow Articles by Atkins, W. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering vol. 9 no. 3 pp. 291-298, 1996
© 1996 Oxford University Press

RESEARCH-ARTICLE

Engineering the aggregation properties of dodecameric glutamine synthetase: a single amino acid substitution controls ‘salting out’

Michael J. Dabrowski, Eric C. Dietze and William M. Atkins1

University of Washington, Medicinal Chemistry Box 357610, Seattle, WA 98195-7610, USA

1To whom correspondence should be addressed

Escherichia coli glutamine synthetase (GS) is a dodecamer of identical subunits which are arranged as two face-to-face hexameric rings. In the presence of 10% ammonium sulfate, wild type GS exhibits a pH-dependent ‘salting out’ with a pKa of 4.51. Electron micrographs indicate that the pH-dependent aggregation corresponds to a highly specific self-assembly of GS tubules, which result from stacking of individual dodecamers. This stacking of dodecamers is similar to the metal ion-induced GS tubule formation previously described. Site-directed mutagenesisexperiments indicate that the N-terminal helix of each subunit is involved in the salting out reaction, as it is in the metal-induced stacking. A single substitution of alanine for His4 completely abolishes the (NH42SO4-induced aggregation. However, the H4C mutant protein does nearly completely precipitate under the same salting out conditions. Mutations at other residues within the helix have no effect on the stacking reaction. Differential catalyticactivity of unadenylylated GS versus adenylylated GS has been used to determine whether wild type dodecamers ‘complement’ the H4A mutant in the stacking reaction. The complementation experiments indicate that His4 residues on bothsides of the dodecamer-dodecamer interfaces are not absolutely required for salting out, although the wild type dodecamers clearly stack preferentially with other wild type dodecamers. Approximately 20% of the protein precipitated fromthe mixtures containing the wild type GS and the H4A mutant is the mutant. The implications of these results for protein engineering are discussed.

Keywords: glutamine synthetase/macromolecular self-assembly/protein aggregation/protein-protein interaction

Received August 30, 1995; revised January 26, 1996; accepted January 30, 1996.


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




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.