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% ␣-helix, 37-40% ␤-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% ␣-helix, 37-46% ␤-structure, and 12-18% random coil for the shark and kidney enzymes. The distribution of intensity between the bands corresponding to protonated and deuteriumexchanged ␣-helices, and between the component bands attributed to ␤-structure, changes considerably on trypsinization, in the direction of a greater proportion of protonated ␣-helix and a broader range of frequencies for ␤-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.

β‐turn formation by a six‐residue linear peptide in solution

“…The complete 1 H NMR assignments of B1 are given in Table 2, and Fig. 4 shows the observed representative cross-peaks in the NOESY experiment, including aN(i, i+2) NOE cross-peak between residues Gly 3 -Tyr 5 , aN(i, i+1) and between residues Asp 4 -Tyr 5 and Gly 3 -Asp 4 , as well as intense NN(i, i+1) NOE cross-peaks between residues Asp 4 -Tyr 5 and Gly 3 -Asp 4 (very close to diagonal peak, Fig. 5).…”

“…Its structural preferences in solution were comprehensively characterized using CD, FT-IR and 1 Understanding the mechanism of secondary structure formation and stability is an essential part of solving the protein-folding puzzle (1)(2)(3). In the past, the structures of protein fragments and designed peptides have been widely explored by spectroscopic analysis, including nuclear magnetic resonance, infrared spectra and circular dichroism measurements, to obtain information on secondary structure formation and stability (4)(5)(6). Occurring as a regular secondary structural element, b-turns are prevalent in globular proteins, and may also be possible sites for nucleation in protein folding.…”

Effects of ELF magnetic field on membrane protein structure of living HeLa cells studied by Fourier transform infrared spectroscopy

“…Several papers have reported the presence of a-helical secondary structure in the membrane transporter or other enzyme proteins [Brazier et al, 1998]. They show that intensities of amide I band are changed with an increase in temperature induced denaturation of enzymes such as (Ca 2þ þ Mg 2þ )-ATPase [Corbalán-García et al, 1994], CaATPase [Jaworsky et al, 1986] and Na, K ATPase [Heimburg et al, 1997]. Also, FT-IR spectroscopy has shown changes in a-helical structure to be a component of the difference between E 1 and E 2 states of Ca 2þ -ATPase and kinetics of folding/unfolding reaction of staphylococal nuclease by FT-IR spectroscopy [Panick et al, 1998].…”

FTIR Study of ATP-Induced Changes in Na+/K+-ATPase from Duck Supraorbital Glands

“…The current data can also be compared with published data for Na 1 /K 1 -ATPase from the shark rectal gland. Heimburg et al (1997) have estimated that ;35% of this enzyme is a-helical. From their data, ;55% of this a-helical structure are in the transmembrane portion of the protein.…”