| We have investigated the dynamic structural mechanism that is responsible for the active Ca{dollar}sp{lcub}2+{rcub}{dollar}-transport by the Ca-ATPase in the skeletal muscle sarcoplasmic reticulum (SR) using time-resolved phosphorescence spectroscopy. We first identified conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase with the phosphorescent probe erythrosin isothiocyanate (ErITC). The major labeling site for ErITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry to be Lys{dollar}sb{lcub}464{rcub}.{dollar} Derivatization with ErITC diminishes the secondary activation of steady-state ATPase activity and the rate of phosphoenzyme decomposition by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations, ErITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of phosphoenzyme decay. Therefore, ErITC modification of the Ca-ATPase does not interfere with the normal catalytic functions associated with the high affinity nucleotide binding site. Furthermore, ATP-dependent activation of the Ca-ATPase is unaffected by detergent solubilization, irrespective of ErITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain.; Using frequency-domain time-resolved phosphorescence spectroscopy, we resolve three rotational correlation times for the Ca-ATPase stabilized in different enzymatic states under equilibrium conditions. These rotational correlation times are assigned respectively to domain motions involving the cytosolic portion of the Ca-ATPase {dollar}(Phisb1approx 5pm 1 mu{dollar}sec), the overall protein rotational motion about the membrane normal {dollar}(Phisb2approx 50pm 10 mu{dollar}sec), and the rotational tumbling of SR vesicles {dollar}(Phisb3approx 1.1pm 0.4{dollar} msec). We find that calcium activation results in an increase in the amplitude of the microsecond domain motions relative to the apoenzyme and phosphoenzyme intermediate, while there are no corresponding alterations in the rates of protein rotational motions or extent of protein-protein interactions. Thus, high affinity calcium binding induces long-range conformational changes that modulate dynamic structural transitions within the ATP hydrolyzing domain, which initiates exclusive phosphoryl transfer from ATP to Asp{dollar}sb{lcub}351{rcub}{dollar} of the Ca-ATPase and facilitates subsequent phosphoenzyme hydrolysis. These results provide direct evidence regarding the dynamic nature of the structural mechanism that couples the free energy released by ATP hydrolysis to the vectorial transport of calcium across the SR membrane. |
