Characterization of the secondary structure and assembly of the transmembrane domains of trypsinized Na,K-ATPase by fourier transform infrared spectroscopy

Abstract

Fourier transform infrared spectroscopy has been used to compare native Na,K-ATPase-containing membranes with those trypsinized in the presence of either Rb+ or Na+ ions to remove the extramembranous parts of the protein. The protein secondary structure content deduced from the amide I band is approximately 30-35% α-helix, 37-40% β-structure, and 13-15% random coil for native membranes from shark rectal gland and from pig kidney, in both the Na- and K-forms. Trypsinization in either RB+ (a K+ congener) or Na+ removes approximately 35% of the amide I band intensity of native membranes from shark rectal gland. The protein secondary structural content of the trypsinized membranes lies in the range of approximately 23-32% α-helix, 37- 46% β-structure, and 12-18% random coil for the shark and kidney enzymes. The distribution of intensity between the bands corresponding to protonated and deuterium-exchanged α-helices, and between the component bands attributed to β-structure, changes considerably on trypsinization, in the direction of a greater proportion of protonated α-helix and a broader range of frequencies for β-structure. The kinetics of deuteration of the slowly exchanging population of protein amide groups is also changed on trypsinization. The mean rate constant for deuteration of trypsinized membranes is approximately half that for native membranes, whereas the proportion of amides contributing to this population increases on trypsinization. The temperature dependence of the amide I band in the Fourier transform infrared spectra indicates that the onset of thermal denaturation occurs at 58°C for native membranes (in either Na+ or K+) and for membranes trypsinized in Rb+, but the major denaturation event for membranes trypsinized in Na+ occurs at approximately 84°C. These results correlate with the functional properties of the intramembranous section of the enzyme.