Label-free methods for optical in vitro characterization of protein-protein interactions.

Fabian Soltermann,Weston B Struwe,Philipp Kukura

doi:10.1039/d1cp01072g

Abstract

Protein–protein interactions are involved in the regulation and function of the majority of cellular processes. As a result, much effort has been aimed at the development of methodologies capable of quantifying protein–protein interactions, with label-free methods being of particular interest due to the associated simplified workflows and minimisation of label-induced perturbations. Here, we review recent advances in optical technologies providing label-free in vitro measurements of affinities and kinetics. We provide an overview and comparison of existing techniques and their principles, discussing advantages, limitations, and recent applications.

Highlights

It is estimated that the human genome codes for more than 500 000 different proteins, of which cells can produce more than 10 000 at any given time.[1]
Given the vital role of proteins in the highly regulated environment of our body, it is not surprising that about 80% of all proteins are expected to function in cooperation with other proteins and substances.[1]
Proteins have to be viewed as part of a complex network of interactions, where changes of one part can induce a cascade of changes in another

Summary

Introduction

It is estimated that the human genome codes for more than 500 000 different proteins, of which cells can produce more than 10 000 at any given time.[1]. Introduced 15 years ago its popularity as a biosensor technology grew rapidly It makes it possible to determine kinetic rate constants and the binding affinities of molecular interactions, without the need for labels.[36] BLI is similar to SPR in the sense that both require immobilization of a ligand on a sensor surface, where analyte binding is detected using an all-optical method (Fig. 2d). In combination with absolute concentration measurements, the average molecular weight and the peak areas of the SEC profile are used to determine the abundance of bound and unbound species, and with that, the binding affinity of protein– protein interactions.[100] Because of the ongoing dilution and separation within the SEC column, true equilibrium is only achieved under carefully chosen conditions, where kinetics are very rapid or very slow on the time scale of the separation.[46] binding affinities obtained under the assumption of fast or slow equilibration should be considered as estimates.[102]. Given the simplicity of the injection and data acquisition procedures, implementation of fully automated and high throughput instruments seems achievable in the near future

Conclusions

Findings

Protein-Protein Interactions