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Biophys Chem. 1996 Apr 16;59(3):341-9.

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

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  • 1Department of Chemistry and Biochemistry, University of California, Los Angeles 90095, USA.


Previous work has revealed phosphate-dependent differences in the complexes formed from myosin subfragment-1 with adenosine diphosphate (S1.ADP) and aluminum fluoride (AlF4-) or beryllium fluoride (BeFx) [Phan and Reisler, Biophys. J., 66 (1994) A78], with the former resembling more the S1**.ADP.Pi 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 nucleotide chase and collisional quenching experiments. epsilon ADP release from S1.epsilon ADP.AlF4- was slower than that from S1.epsilon ADP.BeFx. However, acrylamide titrations of S1.epsilon ADP.AlF4- and S1.epsilon ADP.BeFx 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 SH1 group on S1. To confirm phosphate-related perturbation of the SH1-SH2 sequence, emission spectra of fluorescein (IAF)-labeled SH1 and IANBD-labeled SH2 were recorded for S1 complexes with nucleotides and phosphate analogs. Considerable differences were found 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 opening of the nucleotide cleft on S1 does not change much during ATP hydrolysis [Franks-Skiba et al., Biochemistry, 33 (1994) 12720], while significant changes in the SH1-SH2 region accompany phosphate cleavage.

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