Pearl Cheung

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 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.

Functional studies of yeast actin mutants corresponding to human cardiomyopathy mutations

The molecular mechanisms by which different mutations in actin lead to distinct cardiomyopathies are unknown. Here, actin mutants corresponding to α-cardiac actin mutations causing hypertrophic cardiomyopathy [(HCM) P164A and A331P] and dilated cardiomyopathy [(DCM) R312H and E361G] were expressed in yeast and purified for in vitro functional studies. While P164A appeared unaltered compared to wild-type (WT) actin, A331P function was impaired. A331P showed reduced stability in circular dichroism melting experiments; its monomer unfolding transition was 10°C lower compared to WT actin. Additionally, in vitro filament formation was hampered, and yeast cell cultures were temperature sensitive, implying perturbations in actin–actin interactions. Filament instability of the A331P mutant actin could lead to actomyosin dysfunction observed in HCM. Yeast strains harboring the R312H mutation did not grow well in culture, suggesting that cell viability is compromised. The E361G substitution is located at an α-actinin binding region where the actin filament is anchored. The mutant actin, though unaltered in the in vitro motility and standard actomyosin functions, had a threefold reduction in α-actinin binding. This could result in impairment of force-transduction in muscle fibers, and a DCM phenotype.

Synthetic peptide of the sequence 632-642 on myosin subfragment 1 inhibits actomyosin ATPase activity.

A synthetic peptide corresponding to a sequence 632-642 (S632-642) on the myosin subfragment 1 (S-1) heavy chain and spanning the 50/20 kDa junction of S-1 binds to actin in the presence and absence of S-1. The binding of 1.0 mole of peptide per actin causes almost complete inhibition of actomyosin ATPase activity and only partial inhibition of S-1 binding to actin. The binding of S632-642 to the N-terminal segment of actin is supported by competitive carbodiimide cross-linking of S-1 and S632-642 to actin and the catalytic properties of cross-linked acto-S-1 and actin-peptide complexes. These results show that the sequence 632-642 on S-1 is an autonomous binding site for actin and confirm the catalytic importance of its interactions with the N-terminal segment of actin.

The binding of heat-treated myosin subfragment 1 to actin and substructure considerations

Heat treatment of myosin subfragment 1 at 35 degrees C caused about 95% inactivation of the catalytic function but did not block its binding to actin. Heat-treated subfragment 1 showed specific, strong, and close to stoichiometric binding to actin. MgATP but not MgADP dissociated these complexes. However, in contrast to intact subfragment 1, the heat-treated protein did not polymerize G-actin and was not protected from trypsin by the binding to actin. Tryptic degradation of the 50K fragment abolished, or reduced greatly, the binding of heat-treated subfragment 1 to actin in solution but not on nitrocellulose overlays. These results are discussed in the context of subfragment 1 substructure.