Potential of 13C and 15N 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 IIAmtl 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 15N 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%) 13C 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 13C-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 IIAmtl. This application of 13C 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.
Haris
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PI
Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, London, U.K.
Robillard
G T
GT
van Dijk
A A
AA
Chapman
D
D
D001426:Bacterial Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D003903:Deuterium; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005583:Fourier Analysis; D009004:Monosaccharide Transport Proteins; D009587:Nitrogen Isotopes; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared / Q000379:methods; D014867:Water
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D009587:Nitrogen Isotopes; D014867:Water; D003903:Deuterium; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; C030182:phosphocarrier protein HPr; D017666:Deuterium Oxide