COMPLEXES OF MYOSIN SUBFRAGMENT-1 WITH ADENOSINE-DIPHOSPHATE AND PHOSPHATE ANALOGS - PROBES OF ACTIVE-SITE AND PROTEIN CONFORMATION

Previous work has revealed phosphate-dependent differences in the comp lexes formed from myosin subfragment-1 with adenosine diphosphate (S1 . ADP) and aluminum fluoride (AlF4-) or beryllium fluoride (BeFx) [Pha n 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 .epsilon ADP . AlF4- and S1 .epsilon ADP . BeFx complexes were examined by nucleotid e chase and collisional quenching experiments. epsilon ADP release fro m S1 .epsilon ADP . AIF(4)(-) was slower than that from S1 .epsilon AD P .epsilon BeFx. However, acrylamide titrations of S1 .epsilon ADP . A lF4- and S1 .epsilon ADP . BeFx showed little difference in nucleotide protection from quenching between the two complexes. This contrasts w ith the earlier observation on phosphate analog-dependent changes in t he reactivity of the SH1 group on S1. To confirm phosphate-related per turbation of the SH1-SH2 sequence, emission spectra of fluorescein (IA F)-labeled SH1 and IANBD-labeled SH2 were recorded for S1 complexes wi th nucleotides and phosphate analogs. Considerable differences were fo und between the BeFx and AlF4- complexes with S1 . MgADP for both SH1- and SH2-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 open ing of the nucleotide cleft on S1 does not change much during ATP hydr olysis [Franks-Skiba et al., Biochemistry, 33 (1994) 12 720], while si gnificant changes in the SH1-SH2 region accompany phosphate cleavage.