Monitoring Protein–Ligand Interactions by Time-Resolved FTIR Difference Spectroscopy

Abstract

Time-resolved FTIR difference spectroscopy is a valuable tool to monitor the dynamics and exact molecular details of protein-ligand interactions. FTIR difference spectroscopy selects, out of the background absorbance of the whole sample, the absorbance bands of the protein groups and of the ligands that are involved in the protein reaction. The absorbance changes can be monitored with time-resolutions down to nanoseconds and followed for time periods ranging over nine orders of magnitude even in membrane proteins with a size of 100,000 Da. Here, we discuss the various experimental setups. The rapid scan technique allows a time resolution in the millisecond regime, whereas the step scan technique allows nanosecond time resolution. We show appropriate sample cells and how to trigger a reaction within these cells. The kinetic analysis of the data is discussed. A crucial step in the data analysis is the reliable assignment of bands to chemical groups of the protein and the ligand. This is done either by site directed mutagenesis, where the absorbance bands of the exchanged amino acids disappear or by isotopically labeling, where the band of the labelled group is frequency shifted.