Mapping nucleotide binding site of calcium ATPase with IR spectroscopy: effects of ATP gamma-phosphate binding.
The changes in the IR spectra of the sarcoplasmic reticulum Ca2+-ATPase upon nucleotide binding are recorded in H2O at 1 degrees C in different buffers [imidazole, methylimidazole, 3-(N-morpholino)propanesulfonic acid, and phosphate] at different pH values (pH 6.5-7.8). The difference spectra of nucleotide binding are sensitive to the composition of the solvent. With methylimidazole at pH 7.5 providing the largest binding-induced signals, the effects of gamma-phosphate binding are investigated using ATP, ADP, and beta,gamma-iminoadenosine 5'-triphosphate. The gamma-phosphate contributes approximately 20% to the conformational change seen by IR spectroscopy and affects the beta-sheet structures. The IR experiments also reveal the known affinity difference between ADP and ATP.
Liu
Man
M
Institut für Biophysik, Johann Wolfgang Goethe-Universität, D-60590 Frankfurt am Main, Germany.
Barth
Andreas
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D008956:Models, Chemical; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D013055:Spectrophotometry, Infrared / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014867:Water / Q000737:chemistry
D007093:Imidazoles; D010710:Phosphates; D014867:Water; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; C018100:1-methylimidazole