A Fourier‐Transform Infrared Spectroscopic Investigation of the Hydrogen‐Deuterium Exchange and Secondary Structure of the 28‐kDa Channel‐Forming Integral Membrane Protein (CHIP28)

Abstract

Fourier-transform infrared spectroscopy (FTIR) has been employed to investigate the structural properties of the 28–kDa channel-forming integral membrane protein (CHIP28) present in phospholipid vesicles suspended in aqueous media. This study reports the FTIR spectra of this membrane protein present in H₂O and ²H₂O. The secondary structure of the protein was determined and found to consist of 36%α-helical and 42%β-sheet structures. These results are in close agreement with the results of a previous CD study [Van Hoek, A. N., Wiener, M., Bicknese, S., Miercke, L., Biwersi, J. & Verkman, A. S. (1993) Biochemistry 32, 11847–11856]. However, the results differ from those given in an FTIR analysis by the same workers who recorded FTIR spectra of the CHIP28 protein in a dehydrated state. An unusually high extent of hydrogen-deuterium exchange of the peptide groups of this protein occurs. The magnitude of the spectral changes observed upon exposure of the protein to ²H₂O is greater than has been observed with any other membrane protein previously studied. Thus, over 80% of the peptide groups exchange within 5 min and the amide I band maximum shifts to low frequency by approximately 20cm⁻¹. This high hydrogen-deuterium exchange observed with the CHIP28 protein is consistent with the presence of an aqueous pore within the protein structure.