Investigation of protein–protein interactions by isotope‒edited Fourier transformed infrared spectroscopy

Recent advance in FTIR spectroscopy has shown the usefulness of 13 C uniform isotope labeling in proteins to study protein-protein interactions. 13 C uniform isotope labeling can significantly resolve the spectral overlap in the amide I/Iregion in the spectra of protein-protein complexes, and therefore allows more accurate determination of secondary structures of indi- vidual protein component in the complex than does the conventional FTIR spectroscopy. Only a limited number of biophysical techniques can be used effectively to obtain structural information of large protein-protein complex in solution. Though X-ray crystallography and NMR have been used to provide structural information of proteins at atomic resolution, they are limited either by the ability of protein to crystallize or the large molecular weight of protein. Vibrational spectroscopy, including FTIR and Raman spectroscopies, has been extensively employed to investigate secondary structures and conformational dynamics of protein-protein complexes. However, significant spectral overlap in the amide I/Iregion in the spectra of protein-protein com- plexes often hinders the utilization of vibrational spectroscopy in the study of protein-protein complex. In this review, we shall discuss our recent work involving the application of isotope labeled FTIR to the investigation of protein-protein complexes such as cytokine-receptor complexes. One of the examples involves G-CSF/receptor complex. To determine unambiguously the con- formations of G-CSF and the receptor in the complex, we have prepared uniformly 13 C/ 15 N isotope labeled G-CSF to resolve its amide Iband from that of its receptor in the IR spectrum of the complex. Conformational changes and structural stability of individual protein subunit in G-CSF/receptor complex have then been investigated by using FTIR spectroscopy (Li et al., Bio- chemistry 29 (1997), 8849-8859). Another example involves BDNF/trkB complex in which 13 C/ 15 N uniformly labeled BDNF is complexed with its receptor trkB (Li et al., Biopolymers 67(1) (2002), 10-19). Interactions of 13 C/ 15 N uniformly labeled brain-derived neurotrophic factor (BDNF) with the extracellular domain of its receptor, trkB, have been investigated by em- ploying FTIR spectroscopy. Conformational changes and structural stability and dynamics of BDNF/trkB complex have been determined unambiguously by FTIR spectroscopy, since amide I/Ibands of 13 C/ 15 N labeled BDNF are resolved from those of the receptor. Together, those studies have shown that isotope edited FTIR spectroscopy can be successfully applied to the determination of protein secondary structures of protein complexes containing either the same or different types of secondary structures. It was observed that 13 C/ 15 N uniform labeling also affects significantly the frequency of amide IIband, which may permit the determination of hydrogen-deuterium exchange in individual subunit of protein-protein complexes.