Mapping nucleotide binding site of calcium ATPase with IR spectroscopy: Effects of ATP γ-phosphate binding

The changes in the IR spectra of the sarcoplasmic reticulum Ca²⁺-ATPase upon nucleotide binding are recorded in H₂O at 1°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 γ-phosphate binding are investigated using ATP, ADP, and β,γ-iminoadenosine 5′-triphosphate. The γ-phosphate contributes ∼20% to the conformational change seen by IR spectroscopy and affects the β-sheet structures. The IR experiments also reveal the known affinity difference between ADP and ATP.