Computational protocol: The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II*

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Protocol publication

[…] Fresh S1 prepared after chymotrypsin digestion of myosin was used for steady-state ATPase activity. S1 ATPase activities were determined using [γ-32P]ATP as described (, ). Basal Ca2+-ATPase was determined as per full-length myosin and previously reported for S1 (, ). Both basal Mg2+ and actin-activated Mg2+-ATPase were determined using Mg2+-ATPase buffer without KCl (10 mm imidazole, 0.1 mm CaCl2, 1 mm MgCl2, 1 mm [γ-32P]ATP) as reported previously (). Basal Mg2+-ATPase activities obtained in the absence of actin were subtracted from all actin-activated data points. Actin-activated Vmax and Km values for actin were obtained by fitting all data points from several preparations of wild-type myosin S1 or mutant S1 with the Michaelis-Menten equation using SigmaPlot. Values were averaged to give mean ± S.D. Statistical differences of Ca2+-ATPase, Mg2+-ATPase, Vmax, Km, and catalytic efficiency between wild-type, mutant, and suppressor S1 were carried out using Student's t tests as described previously (). [...] Three-dimensional homology models were generated for the Drosophila wild-type IFI myosin and converter mutant motor domains using the SWISS-MODEL () automatic comparative protein modeling server as described previously (, ). Briefly, the primary sequences of the Drosophila wild-type IFI and converter mutants were aligned pairwise with the sequence of four scallop myosin crystal structures as templates (PDB codes 1KK8, 1QVI, 1S5G, and 1SR6 using the ClustalW alignment protocol, and the alignments were submitted to the alignment interface of SWISS-MODEL (, )). The scallop myosin structures used as templates represent various conformational states of myosin during the cross-bridge cycle; the actin-detached state contains ADP-BeFx (PDB code 1KK8); the pre-power stroke state contains ADP-VO4 (PDB code 1QVI), a conformation that contains partially bound ADP-SO4 (PDB code 1S5G) and the near-rigor state of myosin (PDB code 1SR6), which does not have a nucleotide in the binding pocket. Although scallop templates represent multiple myosin states, they have an alanine at the equivalent position of Asn-509 of Drosophila and may therefore not be the ideal templates to predict the side chain conformation of Asn-509. Chicken smooth muscle myosin has an aspartate at the equivalent position of Asn-509. Therefore, we also used chicken smooth structures as templates to build homology models for wild-type IFI, R759E, and N509K/R759E (PDB codes 1BR1, 1BR2, 1BR4, and 3J04) as smooth chicken myosin and Drosophila IFI share >50% of the myosin head domain sequence. The nucleotide binding pocket of smooth muscle myosin contains MgADP-AlF4 (PDB codes 1BR1 and 1BR2) and MgADP-BeFx (PDB code 1BR4) representing the pre-power stroke state (). The 3J04 template is derived from phosphorylated smooth chicken myosin in the presence of ATP (). Overlay of crystal structures of myosin head domains of scallop (PDB code 1SR6) and Drosophila embryonic myosin (PDB code 4QBD) was done using the Visual Molecular Dynamics (VMD) software (). […]

Pipeline specifications

Software tools SigmaPlot, SWISS-MODEL, Clustal W, VMD
Applications Miscellaneous, Protein structure analysis
Organisms Drosophila melanogaster
Diseases Stroke
Chemicals Adenosine Triphosphate