Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β–barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D) in the absence of actin is completely reversed for each chimera compared to the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared to wild-type IFI, whereas for EMB-3b these values are increased compared to wild-type EMB. In the presence of actin, ADP affinity (KAD) is unchanged for IFI-3a, compared to IFI, but ADP-affinity for EMB-3b is increased, compared to EMB, and shifted towards IFI values. ATP-induced dissociation of acto-S1 (K1k+2) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicate that the exon 3 encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine-binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating acto-myosin cross-bridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms

Identification of functional differences between recombinant human α and β cardiac myosin motors

The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding

Phosphorylation of Ribosomal Protein L18 Is Required for Its Folding and Binding

Ribosomal protein L18 from Bacillus stearothermophilus (bL18) includes a previously unreported phosphoserine residue. The folded conformation of the protein is stabilized by the dianionic form of the phosphate group of that residue. In the absence of Mg , the pK a of the phosphate group is so high that the protein is not fully folded at pH 7. In the presence of Mg , its pK a drops significantly, and consequently the native conformation of bL18 becomes stable at pH 7 and the protein is able to bind to 5S rRNA. Dephosphorylated bL18 does not bind to 5S rRNA at neutral pH

Moderate dietary restriction delays the onset of age-associated sarcopenia in Caenorhabditis elegans due to reduced myosin UNC-54 degradation

Sarcopenia, a gradual decrease in skeletal muscle mass and strength, is a major component of frailty in the elderly, with age, (lack of) exercise and diet found to be the major risk factors. The nematode Caenorhabditis elegans is an important model of sarcopenia. Although many studies describe loss of muscle function in ageing C. elegans, surprisingly few report on the loss of muscle mass. Here, in order to quantify loss of muscle mass under various dietary restriction (DR) conditions, we used an internal GFP standard to determine levels of the major body wall muscle myosin (UNC-54) in transgenic unc-54::gfp worms over their lifespan. Myosin density linearly increased during the first week of adulthood and there was no significant effect of DR. In contrast, an exponential decrease in myosin density was seen during the second week of adulthood, with reduced rates of myosin loss for mild and medium DR compared to control. UNC-54 turnover rates, previously determined using pulse-labelling methods, correspond well with the t1/2 value found here for UNC-54-GFP using fluorescence (control t1/2 = 12.0 days), independently validating our approach. These data indicate that sarcopenia is delayed in worms under mild and medium DR due to a reduced rate of myosin UNC-54 degradation, thereby maintaining protein homeostasis