Biophys J, March 2000, p. 1531-1540, Vol. 78, No. 3

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

Institut für Biophysik, Johann Wolfgang Goethe Universität Frankfurt, D-60590 Frankfurt am Main, Germany

Changes 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 (k_app  3 s¹ in H₂O and k_app  7 s¹ 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 -helical and -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 -phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the -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 1716 cm¹ (H₂O) and 1706 cm¹ (²H₂O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm¹ (H₂O) and 1514 cm¹ (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.

Biophys J, March 2000, p. 1531-1540, Vol. 78, No. 3
© 2000 by the Biophysical Society   0006-3495/00/03/1531/10  $2.00

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