Toshiaki Hiratsuka

Deletion of the Myopathy Loop of Dictyostelium Myosin II and Its Impact on Motor Functions

One of the putative actin-binding sites ofDictyostelium myosin II is the β-strand-turn-β-strand structure (Ile398-Leu-Ala-Gly-Arg-Asp403-Leu-Val405), the “myopathy loop,” which is located at the distal end of the upper 50-kDa subdomain and next to the conserved arginine (Arg397), whose mutation in human cardiac myosin results in familial hypertrophic cardiomyopathy. The myopathy loop contains the TEDS residue (Asp403), which is a target of the heavy-chain kinase in myosin I. Moreover, the loop contains a cluster of hydrophobic residues (Ile398, Leu399, Leu404, and Val405), whose side chains are fully exposed to the solvent. In our study, the myopathy loop was deleted from Dictyostelium myosin II to investigate its functional roles. The mutation abolished hydrophobic interactions of actin and myosin in the strong binding state during the ATPase cycle. Association of the mutant myosin and actin was maintained only through ionic interactions under these conditions. Without strong hydrophobic interactions, the mutant myosin still exhibited motor functions, although at low levels. It is likely that the observed defects resulted mainly from a loss of the cluster of hydrophobic residues, since replacement of Asp403 or Arg402 with alanine generated a mutant with less severe or no defects compared with those of the deletion mutant.

ATP-induced Opposite Changes in the Local Environments around Cys697 (SH2) and Cys707 (SH1) of the Myosin Motor Domain Revealed by the Prodan Fluorescence

To obtain a consistent view of the nucleotide-induced conformational changes around Cys697 (SH2) and Cys707 (SH1) in skeletal myosin subfragment-1 (S-1), the two thiols were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have the same fluorophore at either SH2 or SH1, which was verified by inspections of changes in the ATPases and the localization of fluorescence after tryptic digestion and CNBr cleavage for the two derivatives. Especially, AD was found to be a very useful fluorescent reagent that readily reacts with only SH2 of S-1. Measurements of the nucleotide-induced changes in fluorescence emission spectra of AD-S-1 and BD-S-1 suggested that during ATP hydrolysis the environment around the fluorophore at SH2 is very distinct from that around the fluorophore at SH1, being defined as that the former has the hydrophobic and closed characteristics, whereas the latter has the hydrophilic and open ones. The KI quenching study of the fluorescence of the two S-1 derivatives confirmed these results. The most straightforward interpretation for the present results is that during ATP hydrolysis, the helix containing SH2 is buried in hydrophobic side chains and rather reinforced, whereas the adjacent helix containing SH1 moves away from its stabilizing tertiary structural environment.

Monitoring the Myosin ATPase Reaction Using a Sensitive Fluorescent Probe: Pyrene-Labeled ATP

A pyrene-labeled ATP (Pyr-ATP) in which a pyrene fluorophore is linked to the ribose moiety of ATP with a butyryl chain has been synthesized, together with the corresponding analog of ADP. The spectroscopic properties of two fluorescent analogs were found to be similar to those of 1-pyrenebutyric acid, making them photostable and highly sensitive probes for detecting changes in conformations around the nucleotide binding sites of proteins. Binding of Pyr-ADP to myosin subfragment-1 (S-1) resulted in a fluorescence quenching of about 70%. This binding was tight, with a dissociation constant (0.9 microM) similar to that of ADP itself. Formation of the stable ternary complex of Pyr-ADP with S-1 and orthovanadate could be monitored from the quench in pyrene fluorescence with a rate constant of 0.01 s-1. The final fluorescence intensity was about 20% of that for Pyr-ADP alone. Pyr-ATP was hydrolyzed by S-1 1.3 times faster than was ATP. Hydrolysis of Pyr-ATP was accompanied by an initial quenching of pyrene fluorescence with a subsequent recovery of the fluorescence. The fluorescence changes could be used to monitor the hydrolysis reaction continuously and measure the turnover rates of the analog. The fluorescence assay was sensitive, particularly under single turnover conditions, allowing hydrolysis reactions to be monitored at concentrations of S-1 and the analog as low as 50 nM.

A simple method for the separation of cardiac myosin light chains

A simple and efficient procedure is presented for the separation of pig cardiac myosin light chains. The method employs only three repetitive isoelectric precipitations without column chromatography, and is suitable for use with large quantities of material. The method also permits the recovery of homogeneous light chains with good yields.