Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis
Abstract
Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants. Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset. Suppressor methodology was also applied to the integral membrane protein, diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different. Suppressor as well as sequence co-variation data clearly point to the X-ray structure being the functional one adopted in-vivo. The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists.
Article and author information
Author details
Reviewing Editor
- John Kuriyan, Howard Hughes Medical Institute, University of California, Berkeley, United States
Version history
- Received: June 18, 2015
- Accepted: December 29, 2015
- Accepted Manuscript published: December 30, 2015 (version 1)
- Version of Record published: February 8, 2016 (version 2)
Copyright
© 2015, Sahoo et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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