title: Mapping Protein Binding Sites and Conformational Epitopes Using Cysteine Labeling and Yeast Surface Display.
abstract: We describe a facile method for mapping protein:ligand binding sites and conformational epitopes. The method uses a combination of Cys scanning mutagenesis, chemical labeling, and yeast surface display. While Ala scanning is widely used for similar purposes, often mutation to Ala (or other amino acids) has little effect on binding, except at hotspot residues. Many residues in physical contact with a binding partner are insensitive to substitution with Ala. In contrast, we show that labeling of Cys residues in a binding site consistently abrogates binding. We couple this methodology to yeast surface display and deep sequencing to map conformational epitopes targeted by both monoclonal antibodies and polyclonal sera as well as a protein:ligand binding site. The method does not require purified protein, can distinguish buried and exposed residues, and can be extended to other display formats, including mammalian cells and viruses, emphasizing its wide applicability.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Tariq Ahmad
authors.initials: TA
authors.lastname: Najar
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Shruti
authors.initials: S
authors.lastname: Khare
authors.affiliation: CSIR-Institute of Genomics and Integrative Biology, Mathura Road, New Delhi 110 020, India.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Pandey
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Satish K
authors.initials: SK
authors.lastname: Gupta
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India; Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore 560 064, India. Electronic address: varadar@mbu.iisc.ernet.in.
authors.forename: Raghavan
authors.initials: R
authors.lastname: Varadarajan
mesh_terms: D001665:Binding Sites; D061505:Cell Surface Display Techniques; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016296:Mutagenesis; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D000939:Epitopes; D011506:Proteins; D003545:Cysteine
keywords: cysteine mapping; deep sequencing; high throughput; interaction site; ligand binding; residue burial; saturation mutagenesis; steric repulsion