Polarized infrared attenuated total reflectance spectroscopy of the Ca 2+-ATPase of sarcoplasmic reticulum

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The mean orientations of the transition dipole moments associated with vibrational modes of the proteins and phospholipids of sarcoplasmic reticulum were determined on dry and hydrated membrane multilayers deposited on germanium or zinc selenide crystals, using polarized infrared attenuated total reflectance spectroscopy (P-IR-ATR). For preservation of the enzymatic activity of the Ca 2+-ATPase the films were prepared from solutions containing 0.05 M KCl, 5 mM imidazole (pH 7.4), 0.5 mM MgCl 2, 1–10 mM trehalose and dithiothreitol. The anisotropy was highest in dry films containing ⋍ 7.5 μg protein/cm 2, and sharply decreased with increasing membrane thickness or hydration. The dichroic ratio of the CH 2 vibrations (2923 cm −1) of extracted sarcoplasmic reticulum phospholipids on Ge plate was 1.56, compared with a dichroic ratio of 1.68 obtained on dry films of whole sarcoplasmic reticulum. The dichroic ratios of the amide I band (1650 cm −1) of the Ca 2+-ATPase in the Ca 2-E 1 state and in the EGTA and vanadate stabilized E 2-V state were nearly identical (1.60 vs. 1.62). The dichroism of the amide I, amide II and lipid CH 2 vibrations was not affected by changes in the concentration of KCl (25–100 mM) or Ca 2+ (≃ 10 −8−10 −4M ) and by the addition of vanadate (1 mM) or P 1 (5 mM) in a calcium-free medium containing 0.5 mM EGTA. The dichroic ratio of the C-C (1033 cm −1) or CO stretching band (1046 cm −1) of trehalose incorporated into SR films was 1.2 on Ge plate; this corresponds to a mean angle of ≈ 70° between the plane of the trehalose ring and the normal of the film plane, suggesting that the trehalose molecules are surprisingly well oriented in the polar headgroup region of the phospholipids. The orientation of the trehalose was not affected by the presence of Ca 2+-ATPase.