Use of ¹³C-Labeled Molecules in the Conformational Analysis of Proteins by Ftir Spectroscopy

Abstract

Extensive overlap of the diagnostic IR bands restricts the application of FTIR spectroscopy to the study of conformational changes when two proteins (peptides) form a complex. However, by mixing an isotopically carbon-13 labeled protein with an unlabeled protein or peptide, it is feasible to study their respective conformation-sensitive amide I bands separately [1]. While ¹⁵N labeling causes only subtle changes to the amide I band and to certain side chain absorptions, labeling of proteins with ¹³C results in a 50–55 cm^-1 shift of the amide I band toward lower wavenumbers. We have used this strategy to probe the interaction of calmodulin, a calcium-binding protein (148 amino acids), with small synthetic peptides (22 amino acids) derived from the calmodulin-binding domain of three different target proteins [2]. Calmodulin was uniformly labeled (> 99%) with ¹³C in order to ensure the lack of any remaining intensity arising from calmodulin in the amide I region between 1640–1700 cm^-1, a region in which the relatively weak amide I bands of the target peptides occur. An analysis of the IR spectra revealed that upon binding to calmodulin the conformation of the target peptides changes from an irregular structure to an a-helical conformation. In addition, a slight but perceptible pertubation of the conformation of calmodulin upon binding of the peptides was observed. The isotope-shifting strategy should be more generally applicable to the study of many other protein-peptide and protein-protein interactions, and should also prove useful for studying protein-nucleic acid interactions.