Modification of Loop 1 Affects the Nucleotide Binding Properties of Myo1c, the Adaptation Motor in the Inner Ear

Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotide- and actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, we expressed in insect cells mutants of a truncated form of Myo1c, Myo1c1IQ, as well as chimeras of Myo1c1IQ with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties, or Ca2+ sensitivity of Myo1c1IQ. Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c1IQ-tonic) or replacement with a single glycine (Myo1c1IQ-G) accelerated the release of ADP from A.M 2?3-fold in Ca2+, whereas substitution with loop 1 from phasic muscle myosin II (Myo1c1IQ-phasic) accelerated the release of ADP 35-fold. Motility assays with chimeras containing a single ?-helix, or SAH, domain showed that Myo1cSAH-tonic translocated actin in vitro twice as fast as Myo1cSAH-WT and 3-fold faster than Myo1cSAH-G. The studies show that changes induced in Myo1c via modification of loop 1 showed no resemblance to the behavior of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras

MyBP-C: one protein to govern them all

The heart is an extraordinarily versatile pump, finely tuned to respond to a multitude of demands. Given the heart pumps without rest for decades its efficiency is particularly relevant. Although many proteins in the heart are essential for viability, the non-essential components can attract numerous mutations which can cause disease, possibly through alterations in pumping efficiency. Of these, myosin binding protein C is strongly over-represented with ~ 40% of all known mutations in hypertrophic cardiomyopathy. Therefore, a complete understanding of its molecular function in the cardiac sarcomere is warranted. In this review, we revisit contemporary and classical literature to clarify both the current standing of this fast-moving field and frame future unresolved questions. To date, much effort has been directed at understanding MyBP-C function on either thick or thin filaments. Here we aim to focus questions on how MyBP-C functions at a molecular level in the context of both the thick and thin filaments together. A concept that emerges is MyBP-C acts to govern interactions on two levels; controlling myosin access to the thin filament by sequestration on the thick filament, and controlling the activation state and access of myosin to its binding sites on the thin filament. Such affects are achieved through directed interactions mediated by phosphorylation (of MyBP-C and other sarcomeric components) and calcium

Contractility parameters of human -cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human ?-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human ?-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human ?-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human ?-cardiac myosin contractility as a result of the R403Q mutation

Gender equality and girls education: Investigating frameworks, disjunctures and meanings of quality education

The article draws on qualitative educational research across a diversity of low-income countries to examine the gendered inequalities in education as complex, multi-faceted and situated rather than a series of barriers to be overcome through linear input–output processes focused on isolated dimensions of quality. It argues that frameworks for thinking about educational quality often result in analyses of gender inequalities that are fragmented and incomplete. However, by considering education quality more broadly as a terrain of quality it investigates questions of educational transitions, teacher supply and community participation, and develops understandings of how education is experienced by learners and teachers in their gendered lives and their teaching practices. By taking an approach based on theories of human development the article identifies dynamics of power underpinning gender inequalities in the literature and played out in diverse contexts and influenced by social, cultural and historical contexts. The review and discussion indicate that attaining gender equitable quality education requires recognition and understanding of the ways in which inequalities intersect and interrelate in order to seek out multi-faceted strategies that address not only different dimensions of girls’ and women’s lives, but understand gendered relationships and structurally entrenched inequalities between women and men, girls and boys

Single-molecule imaging reveals how mavacamten and PKA modulate ATP turnover in skeletal muscle myofibrils

Muscle contraction is controlled at two levels: the thin and the thick filaments. The latter level of control involves three states of myosin heads: active, disordered relaxed (DRX), and super-relaxed (SRX), the distribution of which controls the number of myosins available to interact with actin. How these are controlled is still uncertain. Using fluorescently labeled ATP, we were able to spatially assign the activity of individual myosins within the sarcomere. We observed that SRX comprises 53% of all heads in the C-zone compared with 35% and 44% in the P- and D-zones, respectively. The recently FDA-approved hypertrophic cardiomyopathy drug, mavacamten (mava), significantly decreased DRX, favoring SRX in both the C- and D-zones at 60% and 63%, respectively. Since thick filament regulation is in part regulated by the myosin-binding protein-C (MyBP-C), we also studied PKA phosphorylation. This had the opposite effect as mava, specifically in the C-zone where it decreased SRX to 34%, favoring DRX. These results directly show that excess concentrations of mava do increase SRX, but the effect is limited across the sarcomere, suggesting mava is less effective on skeletal muscle. In addition, we show that PKA directly affects the contractile machinery of skeletal muscle leading to the liberation of repressed heads. Since the effect is focused on the C-zone, this suggests it is likely through MyBP-C phosphorylation, although our data suggest that a further reserve of myosins remain that are not accessible to PKA treatment

FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR)

Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I)

The ATPase cycle of human muscle myosin II Isoforms: adaptation of a single mechanochemical cycle for different physiological roles

Striated muscle myosins are encoded by a large gene family in all mammals, including human. These isoforms define several of the key characteristics of the different striated muscle fiber types including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of seven different human myosin isoforms to define the actin.myosin cross-bridge cycle in solution. Here we present data on an eighth isoform the perinatal, which has not previously been charaterized. The perinatal is distinct from the embryonic isoforms appearing to have features in common with the adult fast muscle isoform, including weak affinity of ADP for A.M and fast ADP release. We go on to use a recently developed modeling approach MUSICO to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction including duty ratio, shortening velocity, ATP economy and the load dependence of these parameters. The work shows that the parameters of the cross bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics