SORTCERY - a high-throughput method to affinity rank peptide ligands.

TitleSORTCERY - a high-throughput method to affinity rank peptide ligands.
Publication TypeJournal Article
Year of Publication2014
AuthorsReich LLuther, Dutta S, Keating AE
JournalJournal of molecular biology
Date Publishedoct
ISSN1089-8638
KeywordsBcl-2 family, deep sequencing, FACS, fluorescence-activated cell sorting, high-throughput assay, protein interaction, yeast display
Abstract

Uncovering the relationships between peptide and protein sequences and binding properties is critical for successfully predicting, re-designing and inhibiting protein-protein interactions. Systematically collected data that link protein sequence to binding are valuable for elucidating determinants of protein interaction, but are rare in the literature because such data are experimentally difficult to generate. Here we describe SORTCERY, a high-throughput method that we have used to rank hundreds of yeast displayed peptides according to their affinities for a target interaction partner. The procedure involves fluorescence-activated cell sorting (FACS) of a library, deep sequencing of sorted pools, and downstream computational analysis. We have developed theoretical models and statistical tools that assist in planning these stages. We demonstrate SORTCERY's utility by ranking 1026 BH3 peptides with respect to their affinities for the anti-apoptotic protein Bcl-xL. Our results are in striking agreement with measured affinities for 19 individual peptides with dissociation constants ranging from 0.1 to 60 nM. High-resolution ranking can be used to improve our understanding of sequence-function relationships, and to support the development of computational models for predicting and designing novel interactions.

URLhttp://www.sciencedirect.com/science/article/pii/S0022283614005282
DOI10.1016/j.jmb.2014.09.025
PubMed ID25311858