Complexes of myosin subfragment-1 with adenosine diphosphate and phosphate analogs: probes of active site and protein conformation

Abstract Previous work has revealed phosphate-dependent differences in the complexes formed from myosin subfragment-1 with adenosine diphosphate (S1 · ADP) and aluminum fluoride (AlF 4 − ) or beryllium fluoride (BeF x ) [Phan and Reisler, Biophys. J., 66 (1994) A78], with the former resembling more the S1 ∗ ∗ · ADP · P i state while the latter resembles more the S1 · ATP state. In this work, the conformations of the S1 · eADP · AlF 4 − and S1 · eADP · BeF x complexes were examined by nucleotide chase and collisional quenching experiments. eADP release from S1 · eADP · AlF 4 − was slower than that from S1 · eADP · BeF x . However, acrylamide titrations of S1 · eADP · AlF 4 − and S1 · eADP · BeF x showed little difference in nucleotide protection from quenching between the two complexes. This contrasts with the earlier observation on phosphate analog-dependent changes in the reactivity of the SH 1 group on S1. To confirm phosphate-related perturbation of the SH 1 -SH 2 sequence, emission spectra of fluorescein (IAF)-labeled SH 1 and IANBD-labeled SH 2 were recorded for S1 complexes with nucleotides and phosphate analogs. Considerable differences were found between the BeF x and AlF 4 − complexes with S1 · MgADP for both SH 1 - and SH 2 -labeled proteins. These results are consistent with a recent crystallographic study of S1 complexes with ADP and phosphate analogs [Fisher et al., Biophys. J., 68 (1995) 19S] and the idea that the opening of the nucleotide cleft on S1 does not change much during ATP hydrolysis [Franks-Skiba et al., Biochemistry, 33 (1994) 12 720], while significant changes in the SH 1 -SH 2 region accompany phosphate cleavage.