Quantifying the stabilizing effects of protein-ligand interactions in the gas phase.

Timothy M Allison,Idlir Liko,Arthur Laganowsky,Carol V Robinson,Eamonn Reading,Andrew J Baldwin

doi:10.1038/ncomms9551

Abstract

The effects of protein–ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. Few translate readily to membrane proteins. We have developed an ion-mobility mass spectrometry approach, which discerns ligand binding to both soluble and membrane proteins directly via both changes in mass and ion mobility, and assesses the effects of these interactions on protein stability through measuring resistance to unfolding. Protein unfolding is induced through collisional activation, which causes changes in protein structure and consequently gas-phase mobility. This enables detailed characterization of the ligand-binding effects on the protein with unprecedented sensitivity. Here we describe the method and software required to extract from ion mobility data the parameters that enable a quantitative analysis of individual binding events. This methodology holds great promise for investigating biologically significant interactions between membrane proteins and both drugs and lipids that are recalcitrant to characterization by other means.

Highlights

The effects of protein–ligand interactions on protein stability are typically monitored by a number of established solution-phase assays
These analyses have provided the inspiration for the method implemented in the software presented here, which is based on the work of Hyung et al.[7], where the stability of folded native-like ions, in different bound states, were tracked as a function of collisional activation
Our method provides a novel means of studying the effects of ligand binding on proteins and it is of particular utility when the system under study can bind multiple ligands, such as in the case of oligomeric proteins, where the binding events are challenging to discern using other methods

Summary

Introduction

The effects of protein–ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. By having the collisional activation occur before entry into the mobility cell of the mass spectrometer, the averaged gas-phase collision cross-section (CCS) values (effectively the size) of both folded and unfolded ions can be obtained at a single m/z value, enabling conformational changes to be detected and quantified. These include an approximation of the midpoint between the smallest- and largest-sized species observed, or noting qualitatively different patterns in the unfolding trajectory These analyses have provided the inspiration for the method implemented in the software presented here, which is based on the work of Hyung et al.[7], where the stability of folded native-like ions, in different bound states, were tracked as a function of collisional activation.

Methods

Results

Conclusion