High-throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family.

Márton A Simon,László Nyitray,Péter Ecsédi,Gergő Gógl,Ádám L Póti,József Kardos,Gábor M Kovács,Attila Reményi

doi:10.1111/febs.15175

Abstract

The calcium-binding, vertebrate-specific S100 protein family consists of 20 paralogs in humans (referred as the S100ome), with several clinically important members. To explore their protein-protein interactions (PPIs) quantitatively, we have chosen an unbiased, high-throughput, competitive fluorescence polarization (FP) assay that revealed a partial functional redundancy when the complete S100ome (n=20) was tested against numerous model partners (n=13). Based on their specificity, the S100ome can be grouped into two distinct classes: promiscuous and orphan. In the first group, members bound to several ligands (>4-5) with comparable high affinity, while in the second one, the paralogs bound only one partner weakly, or no ligand was identified. Our results demonstrate that FP assays are highly suitable for quantitative interaction profiling of selected protein families. Moreover, we provide evidence that PPI-based phenotypic characterization can complement or even exceed the information obtained from the sequence-based phylogenetic analysis of the S100ome, an evolutionary young protein family.

Highlights

Biochemical characterization of protein–protein interactions (PPIs) is a challenging field in molecular life sciences, which is usually limited to the determination of steady-state dissociation constants [1]
Competitive fluorescence polarization (FP) as a potent tool to measure highthroughput macromolecular interactions numerous HTP, semiquantitative approaches are available and many low-throughput but highly accurate methods exist to measure PPIs, reliable and quantitative HTP methods are scarce in the literature
Direct FP assay can be performed in large scale in multiwell plates, which makes it an ideal method for rapid interaction screening; it has the serious limitation of chemical labeling that can perturb the binding measurement

Summary

Introduction

Biochemical characterization of protein–protein interactions (PPIs) is a challenging field in molecular life sciences, which is usually limited to the determination of steady-state dissociation constants [1]. The accurate determination of thermodynamic parameters of molecular interactions is performed by fast, but superficial, high-throughput (HTP) methods. Several HTP approaches are applied such as coimmunoprecipitation [2], yeast two-hybrid and spot assays [3], pull-down assay [4], holdup assay [5], and direct fluorescence polarization/anisotropy [6]. In direct fluorescence polarization (FP) experiments, a fluorescent probe (usually a labeled peptide) is titrated with a globular partner. Their association is monitored by the polarization of the emitted light of the fluorophore (Fig. 1A).

Methods

Results

Conclusion