Structural Changes of the Sarcoplasmic Reticulum Ca2+-ATPase upon Nucleotide Binding Studied by Fourier Transform Infrared Spectroscopy

AbstractChanges in the vibrational spectrum of the sarcoplasmic reticulum Ca2+-ATPase upon nucleotide binding were recorded in H2O and 2H2O at −7°C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P3-1-(2-nitrophenyl) adenosine 5′-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca2+-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (kapp ≅ 3s−1 in H2O and kapp ≅ 7s−1 in 2H2O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca2+-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of α-helical and β-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the γ-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the γ-phosphate group in the interactions between the Ca2+-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716cm−1 (H2O) and 1706cm−1 (2H2O) and seem to depend on the charge of the phosphate groups. Bands at 1516cm−1 (H2O) and 1514cm−1 (2H2O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21