POTENTIAL OF C-13 AND N-15 LABELING FOR STUDYING PROTEIN-PROTEIN INTERACTIONS USING FOURIER-TRANSFORM INFRARED-SPECTROSCOPY

In this study, we examine the interaction between two bacterial proteins, namely HPr and IIA(mtl) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy. In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance. Uniform N-15 labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band. However, we show that uniform (90%) C-13 labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band. This opens up windows in different regions of the infrared spectrum. Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly C-13-labeled (in our case HPr), the amide I maxima of this protein shifts by approximately 45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIA(mtl). This application of C-13 labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy. With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible. These could include, among others, enzyme-substrate and protein-ligand interactions.