Mapping Protein Binding Sites and Conformational Epitopes Using Cysteine Labeling and Yeast Surface Display.

Tariq Ahmad Najar, Shruti Khare, Rajesh Pandey, Satish K Gupta, Raghavan Varadarajan

Structure (London, England : 1993) 2017 Mar 07

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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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Citation

Tariq Ahmad Najar, Shruti Khare, Rajesh Pandey, Satish K Gupta, Raghavan Varadarajan. Mapping Protein Binding Sites and Conformational Epitopes Using Cysteine Labeling and Yeast Surface Display. Structure (London, England : 1993). 2017 Mar 07;25(3):395-406