title: 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.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Anusmita
authors.initials: A
authors.lastname: Sahoo
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Shruti
authors.initials: S
authors.lastname: Khare
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Sivasankar
authors.initials: S
authors.lastname: Devanarayanan
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Pankaj C
authors.initials: PC
authors.lastname: Jain
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.|Jawaharlal Nehru Center for Advanced Scientific Research, Bangalore, India.
authors.forename: Raghavan
authors.initials: R
authors.lastname: Varadarajan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D013489:Suppression, Genetic
chemical_list: D001426:Bacterial Proteins; C068356:CcdB protein, Plasmid F; D019852:Diacylglycerol Kinase
keywords: Pairwise contacts; biophysics; model discrimination; none; protein stability; structural biology; structure prediction