Journal Article

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

Harink, Björn, Kortemme, Tanja, Roy, Jagoree, Thorn, Kurt S, Longwell, Scott A, Nguyen, Huy Quoc, Damle, Nikhil P, Baxter, Brian C, Cyert, Martha S, Latorraca, Naomi R, Brower, Kara, Fordyce, Polly Morrell

2019

Overview

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

• Publisher: eLife Science Publications, Ltd,eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Affinity / Algorithms / Amino Acid Sequence / Amino acids / Binding, Competitive / Biochemistry and Chemical Biology / Bioengineering / Biophysics / Calcineurin / Calcineurin - metabolism / Computer applications / Computer simulation / Humans / Hydrogels - chemistry / Immune response / Immunosuppressive agents / Kinases / microfluidics / Microspheres / Models, Theoretical / Mutation / Observations / Peptide Library / Peptide mapping / Peptides / Peptides - metabolism / phophatase specificity / Phosphatases / Phosphoprotein Phosphatases - metabolism / Physiological aspects / Post-translation / Protein Binding / Protein Interaction Maps / Protein phosphatase / Protein Processing, Post-Translational / protein specificity / protein-peptide interactions / Protein-protein interactions / Proteins / Proteins - metabolism / spectral encoding / Structural Biology and Molecular Biophysics / Technology