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. Copyright 2002 Wiley Periodicals, Inc.