Relay Loop Research Articles

Uplink Multiple Access for Reconfigurable Intelligent Surface-Aided Wireless Systems

The integration of reconfigurable intelligent surface- (RIS-) aided wireless communication and multiple access is an attractive and promising scheme for next-generation wireless networks. In this research work, separate uplink RIS-aided nonorthogonal multiple access (NOMA) and uplink relay-assisted NOMA schemes are studied, where the RIS and relay devices are deployed to enhance the coverage of an obstructed single-antenna far user by assisting it to communicate with a single-antenna base station. In each scenario, both perfect successive interference cancellation (pSIC) and imperfect successive interference cancellation (ipSIC) operations are considered in the proposed multiple access network. To characterize the system performance, the associated residual interference caused by ipSIC and relay loop self-interference is characterized using the Rayleigh fading model; subsequently, new channel statistics are derived based on the Gauss-Laguerre polynomial. Consequently, the closed-form approximate outage probability expressions are derived for each scenario in the high signal-to-noise ratio (SNR) regime. To gain further insight, the system throughput in the delay-limited transmission is also obtained for each scenario. The formulated expressions are validated via Monte-Carlo simulations. Finally, the obtained simulation results demonstrate and validate the superiority of the RIS system over the relay device under several system parameters of interest despite the limitation of ipSIC.

A recurrent single-amino acid deletion (p.Glu500del) in the head domain of ß-cardiac myosin in two unrelated boys presenting with polyhydramnios, congenital axial stiffness and skeletal myopathy

BackgroundAlterations in the MYH7 gene can cause cardiac and skeletal myopathies. MYH7-related skeletal myopathies are extremely rare, and the vast majority of causal variants in the MYH7 gene are predicted to alter the rod domain of the of ß-cardiac myosin molecule, resulting in distal muscle weakness as the predominant manifestation. Here we describe two unrelated patients harboring an in-frame deletion in the MYH7 gene that is predicted to result in deletion of a single amino acid (p.Glu500del) in the head domain of ß-cardiac myosin. Both patients display an unusual skeletal myopathy phenotype with congenital axial stiffness and muscular hypertonus, but no cardiac involvement.ResultsClinical data, MRI results and histopathological data were collected retrospectively in two unrelated boys (9 and 3.5 years old). Exome sequencing uncovered the same 3-bp in-frame deletion in exon 15 (c.1498_1500delGAG) of the MYH7 gene of both patients, a mutation which deletes a highly conserved glutamate residue (p.Glu500del) in the relay loop of the head domain of the ß-cardiac myosin heavy chain. The mutation occurred de novo in one patient, whereas mosaicism was detected in blood of the father of the second patient. Both boys presented with an unusual phenotype of prenatal polyhydramnios, congenital axial stiffness and muscular hypertonus. In one patient the phenotype evolved into an axial/proximal skeletal myopathy without distal involvement or cardiomyopathy, whereas the other patient exhibited predominantly stiffness and respiratory involvement. We review and compare all patients described in the literature who possess a variant predicted to alter the p.Glu500 residue in the ß-cardiac myosin head domain, and we provide in-silico analyses of potential effects on polypeptide function.ConclusionThe data presented here expand the phenotypic spectrum of mutations in the MYH7 gene and have implications for future diagnostics and therapeutic approaches.

Filamentous tangles with nemaline rods in MYH2 myopathy: a novel phenotype

The MYH2 gene encodes the skeletal muscle myosin heavy chain IIA (MyHC-IIA) isoform, which is expressed in the fast twitch type 2A fibers. Autosomal dominant or recessive pathogenic variants in MYH2 lead to congenital myopathy clinically featured by ophthalmoparesis and predominantly proximal weakness. MYH2-myopathy is pathologically characterized by loss and atrophy of type 2A fibers. Additional myopathological abnormalities have included rimmed vacuoles containing small p62 positive inclusions, 15–20 nm tubulofilaments, minicores and dystrophic changes. We report an adult patient with late-pediatric onset MYH2-myopathy caused by two heterozygous pathogenic variants: c.3331C>T, p.Gln1111* predicted to result in truncation of the proximal tail region of MyHC-IIA, and c.1546T>G, p.Phe516Val, affecting a highly conserved amino acid within the highly conserved catalytic motor head relay loop. This missense variant is predicted to result in a less compact loop domain and in turn could affect the protein affinity state. The patient’s genotype is accompanied by a novel myopathological phenotype characterized by centralized large myofilamentous tangles associated with clusters of nemaline rods, and ring fibers, in addition to the previously reported rimmed vacuoles, paucity and atrophy of type 2A fibers. Electron microscopy demonstrated wide areas of disorganized myofibrils which were oriented in various planes of direction and entrapped multiple nemaline rods, as corresponding to the large tangles with rods seen on light microscopy. Nemaline rods were rarely observed also in nuclei. We speculate that the mutated MyHC-IIA may influence myofibril disorganization. While nemaline rods have been described in myopathies caused by pathogenic variants in genes encoding several sarcomeric proteins, to our knowledge, nemaline rods have not been previously described in MYH2-myopathy.

Using uniform magnetic field to obtain the constant output power and transfer efficiency for MCR-WPT

Purpose This paper aims to find an approach that achieves constant output power and transfer efficiency in an open space, such as charging pads. Design/methodology/approach In this study, a topology of the five-coil system including two transmitting coils is presented. Also, in a fixed-frequency mode and an open space, this study focuses on the two transmitting coils to achieve the uniform magnetic field and ultimately, attain the constant output power and transfer efficiency. Findings In a fixed-frequency mode and an open space, the constant output power and transfer efficiency is then achieved in experiments by inserting the relay loop into the uniform magnetic field. Practical implications An approach that achieves constant output power and transfer efficiency in an open space. The topology of the five-coil magnetically coupled resonant-wireless power transfer (MCR-WPT) system shows prospective value for various applications, which could be used at designing of wireless battery charger dedicated for cars or mobile phones. Originality/value By comparing the simulation and experimental results, the topology can be optimized in the transmission performance by itself. By doing so, the constant output power and transfer ef?ciency are achieved in the constant frequency mode.

Graded effects of unregulated smooth muscle myosin on intestinal architecture, intestinal motility and vascular function in zebrafish

ABSTRACTSmooth muscle contraction is controlled by the regulated activity of the myosin heavy chain ATPase (Myh11). Myh11 mutations have diverse effects in the cardiovascular, digestive and genitourinary systems in humans and animal models. We previously reported a recessive missense mutation, meltdown (mlt), which converts a highly conserved tryptophan to arginine (W512R) in the rigid relay loop of zebrafish Myh11. The mlt mutation disrupts myosin regulation and non-autonomously induces invasive expansion of the intestinal epithelium. Here, we report two newly identified missense mutations in the switch-1 (S237Y) and coil-coiled (L1287M) domains of Myh11 that fail to complement mlt. Cell invasion was not detected in either homozygous mutant but could be induced by oxidative stress and activation of oncogenic signaling pathways. The smooth muscle defect imparted by the mlt and S237Y mutations also delayed intestinal transit, and altered vascular function, as measured by blood flow in the dorsal aorta. The cell-invasion phenotype induced by the three myh11 mutants correlated with the degree of myosin deregulation. These findings suggest that the vertebrate intestinal epithelium is tuned to the physical state of the surrounding stroma, which, in turn, governs its response to physiologic and pathologic stimuli. Genetic variants that alter the regulation of smooth muscle myosin might be risk factors for diseases affecting the intestine, vasculature, and other tissues that contain smooth muscle or contractile cells that express smooth muscle proteins, particularly in the setting of redox stress.

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 toward wild-type values. The R759E S1 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 that involves 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. Modeling 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.

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

Position of Phenylalanine in the Relay Loop is Important for Myosin Motor Action

We have scanned phenylalanine in the relay loop of Dictyostelium discoideum myosin to study the role of the relay loop in the myosin ATPase actin activation. It is known that C-terminus of the relay loop is on the actin binding interface. Myosin crystal structures show that F506 of the relay loop interacts with F487 of the relay helix in the post-recovery stroke structural state. This interaction is absent in the pre-recovery stroke state due to the linear shift of the relay loop along the relay helix. We hypothesized that actin binding facilitates this conformational change in the relay loop-helix region, thus activating myosin ATPase rate. The goal of the phenylalanine scan was to simulate the loop-helix shift and populate myosin pre- or post-recovery stroke structural states, mimicking proposed effect of actin binding. Two myosin mutants were designed (F506A:D505F and F506A:G507F), along with the wild type myosin (F506) placing phenylalanine in the three consecutive positions within the relay loop. We have used transient time-resolved FRET, myosin intrinsic fluorescence, pyrene labeled actin, and chemical quench to characterize myosin kinetics in detail and conclude on the role of F506 in myosin relay loop. Supported by NIH AR59621.

The mechanism of the converter domain rotation in the recovery stroke of myosin motor protein.

Upon ATP binding, myosin motor protein is found in two alternative conformations, prerecovery state M* and postrecovery state M**. The transition from one state to the other, known as the recovery stroke, plays a key role in the myosin functional cycle. Despite much recent research, the microscopic details of this transition remain elusive. A critical step in the recovery stroke is the rotation of the converter domain from "up" position in prerecovery state to "down" position in postrecovery state that leads to the swing of the lever arm attached to it. In this work, we demonstrate that the two rotational states of the converter domain are determined by the interactions within a small structural motif in the force-generating region of the protein that can be accurately modeled on computers using atomic representation and explicit solvent. Our simulations show that the transition between the two states is controlled by a small helix (SH1) located next to the relay helix and relay loop. A small translation in the position of SH1 away from the relay helix is seen to trigger the transition from "up" state to "down" state. The transition is driven by a cluster of hydrophobic residues I687, F487, and F506 that make significant contributions to the stability of both states. The proposed mechanism agrees well with the available structural and mutational studies.

Interactions between relay helix and Src homology 1 (SH1) domain helix drive the converter domain rotation during the recovery stroke of myosin II

Myosin motor protein exists in two alternative conformations, prerecovery state M* and postrecovery state M**, on adenosine triphosphate binding. The details of the M*-to-M** transition, known as the recovery stroke to reflect its role as the functional opposite of the force-generating power stroke, remain elusive. The defining feature of the postrecovery state is a kink in the relay helix, a key part of the protein involved in force generation. In this article, we determine the interactions that are responsible for the appearance of the kink. We design a series of computational models that contain three other segments, relay loop, converter domain, and Src homology 1 (SH1) domain helix, with which relay helix interacts and determine their structure in accurate replica exchange molecular dynamics simulations in explicit solvent. By conducting an exhaustive combinatorial search among different models, we find that: (1) the converter domain must be attached to the relay helix during the transition, so it does not interfere with other parts of the protein and (2) the structure of the relay helix is controlled by SH1 helix. The kink is strongly coupled to the position of SH1 helix. It arises as a result of direct interactions between SH1 and the relay helix and leads to a rotation of the C-terminal part of the relay helix, which is subsequently transmitted to the converter domain.

Kinetic Characterization of Converter and Relay Loop Domain Interaction in Drosophila Myosin Sub-Fragment 1

We investigated the kinetic properties of Drosophila myosin sub-fragment 1 (S-1) mutant R759E and its suppressor R759E/N509K. The R759E mutation is located in the converter domain of the myosin head whereas N509K is present at the relay loop. Steady-state measurements for R759E S-1 show more than 50% reduction in calcium ATPase as well as ∼40 % reduction in basal magnesium ATPase compared to wild type whereas actin-stimulated Mg-ATPase of R759E (Vmax) is reduced ∼70 %. Homology models of myosin S-1 suggest the R759E mutation can disrupt the interface between the converter and the relay loop and that the suppressor (R759E/N509K) can restore this interaction. This prediction was confirmed by our steady-state ATPase data, which demonstrated that the suppressor mutation restored calcium, basal Mg-ATPase and Vmax back towards wild type values. Calcium and basal Mg-ATPase activity of R759E/N509K increased ∼30 % and ∼25 % respectively compared to mutant R759E S-1. Actin-stimulated Mg-ATPase activity of R759E/N509K was ∼40 % higher compared to R759E mutant S-1. Using flash photolysis, our transient kinetics results showed no significant change in the ATP-induced dissociation of acto-S-1 (K1k+2′) and ADP-affinity (KAD) of acto-S-1 for R759E and R759E/N509K compared to wild-type. Using stopped-flow we measured ATP-binding to S-1 (R759E and R759E/N509K). Compared to wild-type we find for R759E a 30% reduction in the rate constant of ATP-binding (K1k+2) and a 25% reduction in the rate constant of ATP-hydrolysis (k+3+k-3) whereas for R759E/N509K ATP-hydrolysis is restored to wild-type level. This demonstrates the significance of relay-loop and converter domain interaction for S-1's enzymatic ability. The ability to suppress many of the mutant defects in S-1 kinetics corroborates the dramatically improved muscle function we previously found in R759E/N509K double mutants and provides mechanistic insight into this improvement.

Interaction Between the Relay Loop and the SH1-SH2 Helix Region in Drosophila Muscle Myosin is Essential for Normal Motor Function, Myofibril Stability and Muscle Contraction

Molecular modeling of Drosophila muscle myosin reveals that relay domain residue E499 interacts with SH1-SH2 residue R714 in a charge-dependent manner. To explore the significance of this interaction, we generated transgenic lines expressing myosin with a mutation in the relay loop domain (E499R) or the SH1-SH2 helix (R714E). Both mutations yield ∼75% reductions in CaATPase as well as basal or actin-activated MgATPase activity. Actin-sliding velocity of E499R is reduced by 65% and R714E shows no motility. Indirect flight muscles in late pupae of each mutant display disrupted myofibril assembly. Two-hour- and two-day-old adults have severely abnormal myofibril morphology and poor sarcomere organization, with no flight ability. Therefore, the putative interaction of the relay with SH1-SH2 is indispensable for normal motor function, myofibril stability and locomotion. We next constructed a putative compensatory mutant designed to restore function of the E499R or R714E mutant by generating a transgenic line that expresses both E499R and R714E. Interestingly, calcium, basal, and actin-stimulated ATPase values are restored to 65–70% of wild type and actin-sliding velocity is at 40%. The compensatory mutation suppresses the assembly defects in pupal muscle that are seen in both E499R and R714E and reduces disrupted myofibril morphology in two-hour old indirect flight muscles. However, the E499R-R714E mutant myosin was unable to maintain sarcomere organization in two-day-old flies or to restore flight ability. Overall, our results reveal that interaction of residues at the relay/SH1-SH2 helix interface is important for myosin and muscle function.

Disrupting the Myosin Converter-Relay Interface Impairs Drosophila Indirect Flight Muscle Performance

Structural interactions between the myosin converter and relay domains have been proposed to be critical for the myosin power stroke and muscle power generation. We tested this hypothesis by mutating converter residue 759, which interacts with relay residues I508, N509, and D511, to glutamate (R759E) and determined the effect on Drosophila indirect flight muscle mechanical performance. Work loop analysis of mutant R759E indirect flight muscle fibers revealed a 58% and 31% reduction in maximum power generation ( P WL) and the frequency at which maximum power ( f WL) is generated, respectively, compared to control fibers at 15°C. Small amplitude sinusoidal analysis revealed a 30%, 36%, and 32% reduction in mutant elastic modulus, viscous modulus, and mechanical rate constant 2 πb, respectively. From these results, we infer that the mutation reduces rates of transitions through work-producing cross-bridge states and/or force generation during strongly bound states. The reductions in muscle power output, stiffness, and kinetics were physiologically relevant, as mutant wing beat frequency and flight index decreased about 10% and 45% compared to control flies at both 15°C and 25°C. Thus, interactions between the relay loop and converter domain are critical for lever-arm and catalytic domain coordination, high muscle power generation, and optimal Drosophila flight performance.

Functional Mutations in the Force Generation Region Destabilize the Relay Helix in Myosin

We have used pulsed EPR spectroscopy (DEER) to determine the structural effects of point mutations in the force generation region of myosin (I499A, F506A, F692A, D.discoideum sequence). As it was previously reported, these myosin mutants maintain basal ATPase activity, but completely lose their motor function. It was proposed that F506A, located in the relay loop, disrupts communication between the nucleotide binding site and the force generation region in myosin, and F692A and I499A, located on the interface with the converter domain, affect coordination of the converter domain relative to the myosin head. In this study we assayed the structure of the relay helix in these mutants and in wild-type myosin. The mutations were introduced into the A639C:K498C myosin construct, both Cys were labeled with maleimide spin probes, and interprobe distance was measured. Previous studies showed that probes at these sites detect the nucleotide-induced bending of the relay helix. Observed spin echo decays were interpreted in terms of one or two Gaussian distance distributions, corresponding to one or two myosin structural states. Nucleotide analogs were used to trap myosin in several biochemical states. In the wild type, two structural states, corresponding to pre- and post-recovery stroke, are present in a single biochemical state. The mutations affect the equilibrium between these two structural states and increase the conformational disorder, indicating that these mutants destabilize the structure of the force-generation region, particularly in the post-recovery state. These results provide a structural explanation for the functional perturbation, introduced to myosin.

Mutating the Converter–Relay Interface of Drosophila Myosin Perturbs ATPase Activity, Actin Motility, Myofibril Stability and Flight Ability

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase ( V max) by ∼ 60% compared to wild-type myosin, but there is no change in apparent actin affinity ( K m). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by ∼ 15% or ∼ 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by ∼ 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional “cracking” of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.

Converter Domain Residue R759 Interaction with Relay Loop Residue N509 in Drosophila Muscle Myosin is Critical for Motor Function, Myofibril Stability and Flight Ability

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of Drosophila melanogaster skeletal muscle myosin. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at the relay loop interaction site. The mutation depresses calcium, basal or actin-activated MgATPase values (Vmax) by ∼60% and actin sliding velocity ∼35% compared to wild-type myosin. Ultrastructure of two-day-old adult fibers shows cracking and frayed myofibrils with some disruption of the myofilament lattice which becomes more severe in one-week-old adults. Flight ability is reduced in two-day-old flies compared to controls and is absent in 1-week-old adults. Thus appropriate interaction between the relay loop and converter domain is essential for normal motor function, myofibril stability and locomotion. To examine the specificity of this interaction, we used a compensatory mutational approach to attempt to restore the function of the R759E mutant myosin. Our modeling indicates that relay loop residues N509 and D511 interact with converter domain residue R759. To verify our model, we generated two transgenic lines that express R759E and either the N509K or D511K mutations. Interestingly, calcium, basal, and actin stimulated ATPase values are restored to 70% and actin sliding velocity is restored to 90% in N509K/R759E but not in D511K/R759E. Structurally fibers from 2-day or one-week -old adults appear morphologically normal in N509K/R759E and their flight ability is like wild type. However, D511K/R759E myofibrils do not show any improvement compared to R759E and flight ability is worse than R759E. Overall, our results reveal the critical interaction between the converter domain with relay loop residues and their role in myosin motor function and myofibril assembly/stability.

Relay Loop Stabilizes the Force-Generating Region in Myosin

We have used transient time-resolved FRET (TR2FRET) to monitor the conformation of the relay helix in a myosin II functional mutant during the recovery stroke in real time. Myosin was perturbed with the F506A mutation (Dictyostelium discoideum sequence), located within the relay loop in the force-generating region. F506 is a highly conserved residue in myosin II and is a hypertrophic cardiomyopathy mutation site. Previous studies [Tsiavaliaris, EMBO Rep, 2002, 3(11), 1099] showed a significant effect of the F506A mutation on myosin function. Actin affinity in the presence of ATP was increased, and the mutant did not move actin filaments in in vitro motility assays. A small decrease in intrinsic fluorescence was observed upon addition of excess ATP, but ATP binding and hydrolysis were not affected by the mutation. It was proposed that the F506A disrupts the communication between the active site and the lever arm. We engineered a double-Cys myosin mutant (A639C:K498C) in the Cys-less background with the F506A functional mutation, and labeled the mutant with optical probes. We used TR-FRET to determine the interprobe distance, and TR2FRET measurements after rapid mixing with ATP revealed changes in the relay helix conformation during the recovery stroke in real time. The mutation induced significant disorder of the relay helix in the force-generating region, but myosin still produces a recovery stroke, changing the relay helix conformation from straight to bent. We conclude that (a) the relay helix is disordered in myosin functional mutant F506A, which demonstrates the importance of the relay loop - relay helix interaction in the relay helix stabilization, and (b) the relay helix is the major structural element in the force-generating region of myosin, responsible for communication from the active site to the converter domain and the lever arm.

Adaptive relay-based control of household freezers with on–off actuators

This paper describes an adaptive scheme for temperature control in household freezers with low-end sensing and actuation equipment, like on–off compressors and dampers. The scheme is based on an auxiliary filter introduced in the relay loop to alter and improve the characteristics of the induced oscillation. The filter is tuned adaptively to cope with system variability and uncertainty. Some tests on detailed simulation models of a commercial appliance are reported to validate the approach and show its effectiveness with respect to both food preservation and energy consumption requirements.

Disrupting Myosin Relay-Converter Domain Communication Impairs Drosophila Muscle Mechanical Performance and Flight Ability

We evaluated the mechanical properties of Drosophila indirect flight muscle (IFM) fibers expressing a myosin converter domain R759E mutation. The interaction of R759 with relay loop residue I508 is thought to be critical for relay-converter inter domain communication. By changing the charge on residue 759, we are testing if this inter domain interaction is important for the mechanical performance of muscle fibers. Electron microscopic examination of muscle fibers from young adult R759E flies indicates normal myofibril assembly. Using the work loop analysis technique we found that the maximum power (Pmax) generated by the mutant R759E fibers from two day old flies was significantly reduced by 50% compared to control fibers while the frequency at which maximum power is generated (fmax) was reduced to 67%. Maximum power occurred at peak-to-peak strain amplitude of 2% resting muscle fiber length. Varying ATP concentration at 15°C revealed no significant difference in Km for Pmax or fmax between control and mutant R759E fibers, suggesting that the mutation does not affect ATP affinity. Small amplitude sinusoidal analysis revealed a significant reduction in complex stiffness by 48% compared to control fibers, with elastic modulus, Ee, reduced by 31% and viscous modulus, Ev, reduced by 45%. This reduction in power and mechanical performance of the flight muscle fibers led to a decrease in wing beat frequency from 140 ± 2 Hz for control flies to 127 ± 2 Hz. The reduction in wing beat frequency contributed to a decrease in flight index from 2.31 ± 0.1 for control flies to 1.25 ± 0.1 at 15°C. Thus, this study suggests that the interaction between relay loop I508 and converter domain R759 is critical for myosin inter domain communication, muscle fiber power generation and Drosophila flight performance.

Kinetic Characterization of the Function of Myosin Loop 4 in the Actin−Myosin Interaction

Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (DeltaAL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.