The relay-converter interface influences hydrolysis of ATP by skeletal muscle myosin II

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1 we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase towards wild-type values. The S1-R759E construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke and involving a change in conformation at the relay/converter domain interface. Significantly the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modelling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine tuning myosin kinetics, in particular ATP binding and hydrolysis

Modeling the Actin.myosin ATPase cross-bridge cycle for skeletal and cardiac muscle myosin isoforms

Modeling the complete actin.myosin ATPase cycle has always been limited by the lack of experimental data concerning key steps of the cycle, because these steps can only be defined at very low ionic strength. Here, using human ?-cardiac myosin-S1, we combine published data from transient and steady-state kinetics to model a minimal eight-state ATPase cycle. The model illustrates the occupancy of each intermediate around the cycle and how the occupancy is altered by changes in actin concentration for [actin] = 1–20Km. The cycle can be used to predict the maximal velocity of contraction (by motility assay or sarcomeric shortening) at different actin concentrations (which is consistent with experimental velocity data) and predict the effect of a 5 pN load on a single motor. The same exercise was repeated for human ?-cardiac myosin S1 and rabbit fast skeletal muscle S1. The data illustrates how the motor domain properties can alter the ATPase cycle and hence the occupancy of the key states in the cycle. These in turn alter the predicted mechanical response of the myosin independent of other factors present in a sarcomere, such as filament stiffness and regulatory proteins. We also explore the potential of this modeling approach for the study of mutations in human ?-cardiac myosin using the hypertrophic myopathy mutation R453C. Our modeling, using the transient kinetic data, predicts mechanical properties of the motor that are compatible with the single-molecule study. The modeling approach may therefore be of wide use for predicting the properties of myosin mutations

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

Dilated cardiomyopathy myosin mutants have reduced force-generating capacity

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human ?-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M.D complex in the steady-state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force holding capacity due to the reduced occupancy of the force-holding state

Distribution-level power electronics : soft open-points

This thesis considers the use of medium-voltage power electronic compensation at distribution network voltage levels (11kV) for the improvement of power quality, reliability, and to accommodate growth in customer demand or distributed generation capacity. Specifically, power electronic compensators connecting two or more nodes of previously isolated radial feeders are considered. This type of device can be considered as an alternative to normally-open points, which connect two nodes with mechanical switchgear. Rather than connecting these nodes directly, power-electronics are placed between them. This type of device will be deemed a soft-open point (SOP) in this thesis. Several compensator topologies which can achieve the functionality of a SOP are considered. The feature criteria used to choose which compensators are suitable for use as a SOP are: the ability to transfer active power between feeders; the ability to resupply (post-fault) adjacent feeders connected via the compensator; an inherent or controlled disturbance rejection or fault current limiting between adjacent feeders. Modified versions of some existing flexible AC transmission system (FACTS) or custom power devices o er the potential to meet these criteria. The compensator topologies considered include: static synchronous series compensators, unified power flow controllers, back-to-back connected voltage-sourced converters (VSCs) or multi-terminal connected VSCs. In order to quantify and compare the benefits of these compensator topologies, their relative performance on UK distribution networks is assessed based on load flow and optimal power flow case studies performed on datasets representing several hundred UK distribution networks. Benefits quantified include an increase in customer reliability ratings, prevention or deferral of asset replacement, reduction in conductor losses, accommodation of increased distributed generation, and accommodation of increased customer demand. The benefit analyses show that multi-terminal VSC based SOPs provide the greatest flexibility, but one must recognize that associated cost and right-of-way issues associated with distribution networks can be prohibitive. Series and series-shunt compensators are shown to offer an an adequate amount of control, achieving reasonable levels of load and generation growth with lower overall estimates for cost. Several control strategies and converter topologies are considered for use in SOP implementation under a number of scenarios. The use of multi-terminal VSCs is also verified through implementation in a prototype network and through time-domain simulations. These demonstrations serve as a proof of concept for SOP operation in scenarios relevant to their intended use in distribution networks. Also considered is the use of SOPs to directly compensate overload substation transformers, for which it is found that SOPs can very effectively mitigate overload events at the expense of increased cumulative losses. Different high-level control schemes are shown to reduce the impact of the additional converter losses