Monitoring protein-ligand interactions by time-resolved FTIR difference spectroscopy.
Time-resolved FTIR difference spectroscopy is a valuable tool to monitor the dynamics of protein-ligand interactions, which selects out of the background absorbance of the whole sample the absorbance bands of the protein groups and of the ligands, which are involved in the protein reaction. The absorbance changes can be monitored with time-resolutions down to nanoseconds and followed then over nine orders of time up to seconds even in membrane proteins with the size of 100,000 Dalton. Here, we will discuss the various experimental setups. We will show new developments for sample cells and how to trigger a reaction within these cells. The kinetic analysis of the data will be discussed. A crucial step in the data analysis is the clear-cut band assignment to chemical groups of the protein and the ligand. This is done either by site directed mutagenesis or by isotopically labeling. Examples for band assignments will be presented in this chapter.
Kötting
Carsten
C
Department of Biophysics, Ruhr-University Bochum, Bochum, Germany.
Gerwert
Klaus
K
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D013997:Time Factors
D008024:Ligands; D011506:Proteins; D001436:Bacteriorhodopsins