RRC ID 53671
Author Sahoo A, Khare S, Devanarayanan S, Jain PC, Varadarajan R.
Title Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis.
Journal Elife
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.
Volume 4
Published 2015-12-30
DOI 10.7554/eLife.09532
PII e09532
PMID 26716404
PMC PMC4758949
MeSH Bacterial Proteins / chemistry* Bacterial Proteins / genetics Diacylglycerol Kinase / chemistry* Diacylglycerol Kinase / genetics Escherichia coli / chemistry Escherichia coli / enzymology* Escherichia coli / genetics Models, Molecular Mutagenesis* Protein Conformation Suppression, Genetic*
IF 7.08
Times Cited 13
Prokaryotes E. coli