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

Abstract

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.

MRBLES 2.0: High-throughput generation of chemically functionalized spectrally and magnetically-encoded hydrogel beads using a simple single-layer microfluidic device

“…Comparison of fluorescence signals for beads polymerized in the presence of 2 different concentrations of 10-UA reveal that intensities are higher but slightly more heterogeneous for 0.065% (v/v) 10-UA, likely reflecting incomplete coverage of the whole droplet surface 32 . Estimation of loading densities by DNA conjugation for the 0.2% (v/v) 10-UA condition suggest ∼10 7 to 10 8 oligos bind to the surface of each bead, consistent with prior estimates of MRBLEs peptide loading capacities 30 .…”

“…In prior work, -COOH or -NH 2 moieties were covalently coupled to the PEG-DA polymer chains comprising the MRBLEs hydrogel matrix via Michael addition after bead synthesis 30 . However, this method was relatively slow (requiring ∼24-48 hours) and susceptible to inhomogeneous distribution of free acrylate groups on bead surface, leading to heterogeneity of coverage during coupling.…”

“…Lns have narrow and well-separated emission spectra, making it theoretically possible to generate code sets with 10 5 -10 6 unique members 28 , and we previously demonstrated the ability to produce and discriminate > 1,100 codes 27 . In addition, MRBLEs can be subsequently functionalized for on-bead solid-phase peptide synthesis 29,30 . However, prior MRBLEs production pipelines were complex, requiring two-layer microfluidic devices with integrated valves and extensive custom pneumatics control hardware 27,28 , limiting widespread adoption.…”

“…Using the MRBLEs 2.0 pipeline, we demonstrate the ability to generate monodisperse MRBLEs (CV<7%) containing 48 distinct well-resolved spectral codes (<0.01% probability of code-misassignment) in high-throughput (>∼3,000,000 beads with diameter ∼50 μm within 20 min per code, including washing steps). To highlight the utility of these MRBLEs in downstream assays, we conjugate amine-functionalized oligonucleotides and entire proteins to MRBLEs bearing carboxyl groups with estimated bead loading densities of 10 7 -10 8 molecules/bead, and additionally conjugate biotin molecules to amine-functionalized MRBLEs, demonstrating the feasibility of direct on-bead peptide synthesis 29,30 . To facilitate bead separation and washing during assays, we additionally show that spectrally encoded beads can also be magnetized via simultaneous incorporation of magnetic nanoparticles with little effect on spectral codes.…”

High-throughput binding affinity measurements for mutations spanning a transcription factor-DNA interface reveal affinity and specificity determinants

“…S5), allowing reliable determination of mutations that both increased and decreased affinity relative to wildtype Pho4. To estimate affinities for deleterious variants with Kd values above concentrations probed experimentally, we globally fit all binding curves to a Langmuir isotherm with a single shared saturation value and individually-fit Kd values (Fordyce, et al, 2010;Fordyce, et al, 2012;Nguyen, et al, 2019). Extracted Kd and ΔΔG values for all mutants across experiments were highly reproducible over an energetic range of ~4 kcal/mol (Kd: r 2 = 0.96, RMSLE = 0.22 nM; ΔΔG: r 2 = 0.96, RMSE = 0.31 kcal/mol) (Figs.…”