Myosin Motor Domain Research Articles

Mammalian Myosin-18A, a Highly Divergent Myosin

The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 μm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s(-1)) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.

Analysis of Myosin Motor Domain Interactions with its Chaperone UNC-45 at the Single Molecule Level

Myosins are actin-based motor proteins that convert chemical energy from ATP hydrolysis into mechanical work. Movement of myosin heads along actin filaments is a result of structurally complex conformational changes in the myosin motor domain induced by ATP binding and hydrolysis. UNC-45, a member of the UCS family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanism of the myosin-UNC-45 interaction in the promoting proper folding of the myosin head domain is not known. We have devised a novel approach to elucidate the interaction of the UNC-45 chaperone with the myosin motor domain utilizing single molecule atomic force microscopy (AFM). By chemically coupling a titin I27 polyprotein to the motor domain of myosin we synthesized a chimera protein that possesses the property of a “molecular reporter”. Our new construct provides a specific attachment point and the well-characterized mechanical fingerprint of the titin octamer in the AFM measurements. Refolding experiments of the chimeric S1-I27 molecules showed that the myosin motor domain interfered with the refolding of otherwise robust I27 modules, presumably by recruiting them into a misfolded state. The presence of UNC-45 restored the folding of the titin I27 domains. We identify the canonical UCS domain of UNC-45 as the essential component of chaperone like activity. This approach enables the study the myosin-UNC-45 interactions at a single molecule level and their consequences for motor domain folding and misfolding in mechanistic detail.

Ultrafast Force-Clamp Spectroscopy of Single Molecular Motors and DNA Binding Proteins

Force plays a fundamental role in a wide array of biological processes, regulating, for example, enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. Here, we present a novel force-clamp laser trap that is capable of applying constant loads to molecular complexes with lifetimes above ∼10 μs [Capitanio et al., Nature Methods 9, 1013-1019 (2012)]. Such capability is enhanced by a detection strategy allowing the investigation of very short interactions (∼100μs). Moreover, the high temporal and spatial resolution of the method enables us to probe sub-nanometer conformational changes with a time resolution of few tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. These results resolve the molecular mechanism underlying regulation of muscle contraction by force. Our technique could be straightforwardly applied to a wide variety of non-processive molecular motors, single domains of processive motors, protein-DNA and protein-RNA interactions, and conceivably to any short-lived protein-protein interaction, opening new avenues for investigating the effects of forces on biological processes.

Coordination of Actin-Activated Phosphate Release and the Myosin Power-Stroke Detected by Transient Time-Resolved FRET

We have used transient kinetics and nanosecond time-resolved FRET to resolve a structural transition in the myosin relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with force-transducing rotation of the myosin light chain domain. Previous research in the absence of actin used optical and EPR spectroscopy to demonstrate that the relay helix of Dictyostelium Discoideum myosin II exists in dynamic equilibrium between a bent pre-power-stroke state, stabilized by ATP, and a straight post-power-stroke state that predominates when ADP or no nucleotide is bound. Kinetics studies showed that ATP binding induces a recovery stroke in which this equilibrium is shifted toward the bent state. In the present study, we ask whether actin binding to the myosin.ADP.Pi complex reverses this transition and if so, how actin-activated helix straightening is coordinated with biochemical transitions of the myosin ATPase cycle. We labeled a Cys-lite Dictyostelium myosin motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We measured time-resolved FRET following stopped-flow mixing of myosin (in the presence of ATP) with actin. These experiments allowed us to measure the structural kinetics of the relay helix during the weak-to-strong actin-binding transition. The results clearly show that actin binding straightens the relay helix in the presence of ATP. The helix straightens prior to ADP dissociation and full formation of the strong binding actomyosin interface. Measurements of actin-activated phosphate release demonstrate that dissociation of the hydrolyzed gamma-phosphate is closely coupled to the structural state of the relay helix during the power-stroke.

Cloning, Characterization, and Promoter Analysis of Mouse Myo10 Gene

Myosin X (Myo10) is an unconventional myosin associated with filopodia motility. Recent studies show that in addition to full-length Myo10, brain expresses a shorter form of Myo10 that lacks a myosin motor domain named headless Myo10. Herein, we analyzed and cloned 2-kb of the 5′-upstream sequences of mouse full-length Myo10 (fMyo10) and headless Myo10 (hMyo10) to understand the transcriptional regulation of the Myo10 gene. The putative transcription factor binding sites and CpG island were analyzed by a bioinformatic approach. Luciferase reporter assays showed that the 2-kb of 5′-upstream sequences of both fMyo10 and hMyo10 had promoter activities.

Whole genome duplication events in plant evolution reconstructed and predicted using myosin motor proteins

BackgroundThe evolution of land plants is characterized by whole genome duplications (WGD), which drove species diversification and evolutionary novelties. Detecting these events is especially difficult if they date back to the origin of the plant kingdom. Established methods for reconstructing WGDs include intra- and inter-genome comparisons, KS age distribution analyses, and phylogenetic tree constructions.ResultsBy analysing 67 completely sequenced plant genomes 775 myosins were identified and manually assembled. Phylogenetic trees of the myosin motor domains revealed orthologous and paralogous relationships and were consistent with recent species trees. Based on the myosin inventories and the phylogenetic trees, we have identified duplications of the entire myosin motor protein family at timings consistent with 23 WGDs, that had been reported before. We also predict 6 WGDs based on further protein family duplications. Notably, the myosin data support the two recently reported WGDs in the common ancestor of all extant angiosperms. We predict single WGDs in the Manihot esculenta and Nicotiana benthamiana lineages, two WGDs for Linum usitatissimum and Phoenix dactylifera, and a triplication or two WGDs for Gossypium raimondii. Our data show another myosin duplication in the ancestor of the angiosperms that could be either the result of a single gene duplication or a remnant of a WGD.ConclusionsWe have shown that the myosin inventories in angiosperms retain evidence of numerous WGDs that happened throughout plant evolution. In contrast to other protein families, many myosins are still present in extant species. They are closely related and have similar domain architectures, and their phylogenetic grouping follows the genome duplications. Because of its broad taxonomic sampling the dataset provides the basis for reliable future identification of further whole genome duplications.

A Structural Model of Actomyosin in the Prepowerstroke State: Actin Binding Induced Extra Priming of Myosin

The effective step of myosin's working cycle is the powerstroke. Although, there are atomic structural models of the actomyosin complex in rigor state, there is a great demand for atomic models of the complex in the prepowerstroke state as well, in order to understand the mechanism of the powerstroke. As the actin bound prepowerstroke state of myosin is experimentally challenging to capture because of its short life-time and low proportion of the cycle, first we created an in silico structural model of such state. We docked the relaxed structures of myosin in the weak actin binding and up lever state to an actin trimer, then relaxed the complex. Interestingly, actin binding induced a further up movement of the lever accompanied with a further closure of the switch II loop in a nanosecond time-scale. This state may represent an ‘extra primed' state of myosin that precedes the powerstroke. Additionally, when the interaction between activation loop and actin N-terminal region was interrupted in the docked and relaxed actomyosin by applying a mutation, myosin relaxed back to its original up lever conformation. In order to test our in silico results, we experimentally examined the lever movement upon actin binding. GFP and ReAsH were introduced into the C- and N-terminal of Dictyostelium myosin motor domain to follow the lever position by FRET. We conclude that in silico and experimental results reveal a new state of actomyosin at the beginning of the powerstroke, indicating the initial mechanism of actin activation of myosin.

Lack of Developmental Redundancy between Unc45 Proteins in Zebrafish Muscle Development

Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p) protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b−/−; unc45a−/− mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b−/− and unc45b−/−; unc45a−/− embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 μs, allows detection of interactions as brief as ∼100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

Tracking UNC-45 Chaperone-Myosin Interaction with a Titin Mechanical Reporter

Myosins are molecular motors that convert chemical energy into mechanical work. Allosterically coupling ATP-binding, hydrolysis, and binding/dissociation to actin filaments requires precise and coordinated structural changes that are achieved by the structurally complex myosin motor domain. UNC-45, a member of the UNC-45/Cro1/She4p family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach, to our knowledge, to analyze the interaction of UNC-45 with the myosin motor domain at the single molecule level using atomic force microscopy. By chemically coupling a titin I27 polyprotein to the motor domain of myosin, we introduced a mechanical reporter. In addition, the polyprotein provided a specific attachment point and an unambiguous mechanical fingerprint, facilitating our atomic force microscopy measurements. This approach enabled us to study UNC-45–motor domain interactions. After mechanical unfolding, the motor domain interfered with refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach enables the study of UNC-45 chaperone interactions with myosin and their consequences for motor domain folding and misfolding in mechanistic detail.

A New Approach for the Identification of Allosteric Binding Sites in Proteins

Recent results indicate that small‐molecule‐protein interactions modulate the function of a large fraction of the enzyme complement in eukaryotic cells. In particular, modulation of enzyme functions by small metabolites that are not their substrates or products is increasingly recognized as a mechanism by which cells can adapt to changes in environmental conditions. While the effect of metabolite‐binding on conformational transitions and thus protein folding, stability, and catalytic activity is clearly of considerable importance, studying metabolite‐protein interactions is in most instances impeded by the low‐affinity nature of the interactions, their moderate effect on enzyme activity, and the complex nature of their interference with substrate turnover. Here, we describe an approach for the efficient mapping of the binding sites of nutrient polyphenols and small dye molecules to the 80 kDa myosin motor domain. We speculate that their binding occurs preferentially with sites that are important for protein‐metabolite interactions and can also be exploited as binding sites for therapeutic drugs. To determine the affinity of the prolific binders for the myosin‐2 motor domain and to select the most suitable compounds, we performed microscale thermophoresis experiments. Subsequently, we determined the structure of the binding pockets for the most promising compounds by X‐ray crystallography.

Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A

AbstractWe have generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with green fluorescent protein was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5, whereas homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction, and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live-cell imaging of megakaryocytes in the BM show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most probably in humans. We also observed premature cataract formation, kidney abnormalities, including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domain manifest similar hematologic, eye, and kidney phenotypes to humans with MYH9-related disease.

Structural Model of the Pre-Powerstroke State of the Actomyosin Complex

We created a structural model of the ADP.Pi pre-powerstroke state of Dictyostelium myosin motor domain complexed with actin trimer by an in silico protein docking procedure followed by a long timescale molecular dynamics relaxation. Furthermore, we also modeled the ADP.Pi down lever state of myosin motor domain and the rigor complexes using 1q5q (apo structure of Dictyostelium myosin motor domain) or squid myosin S1 (3I5G) atomic structures and actin trimer model. During the molecular dynamics of the apo myosin (1q5q ) complexed with actin trimer the actin binding cleft of myosin motor domain spontaneously closed and the relaxed actomyosin rigor structure fits well with the structural rigor model determined by EM. The analysis of the ADP.Pi pre-powerstroke actomyosin complex showed that actin spontaneously induces significant conformational changes in the myosin motor domain. Most strikingly, actin further closes the closed switch 2 loop coupled with a further up movement of the lever. Interestingly, if the interaction between the N-terminal region of actin and myosin activation loop is interrupted by a single mutation (K520N) this conformational change does not occur upon actin binding. Furthermore, actin binding rearranges correlating movements of different myosin motor regions which effect was significantly reduced in the mutant. Recently we shoved experimentally that the interaction between actin and the activation loop is responsible for channeling the enzymatic pathway of actomyosin into the effective powerstroke path. The further closure of switch 2 induced by actin indicates that cocking is induced by actin mainly through the actin binding of activation loop. In order to test the indicated conformational change we have produced a myosin motor domain containg a FLASH and REASH probes located at the N and C-terminus, respectively, to sensitively follow the lever movement upon actin binding by FRET.

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover activity of myosin in tarantula leg-muscle fibers by observing single turnovers of the fluorescent nucleotide analog, mantATP, as monitored by the decrease in fluorescence when mantATP is replaced by ATP in a chase experiment. We find a multi-exponential process, with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant, ∼30 minutes. This slow turnover state is termed the super-relaxed state (SRX) and is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state (days) employing a lie-in-wait hunting strategy. If fibers are incubated in mantADP and chased with ADP, the SRX is not seen, indicating that relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with the very long lifetime. The presence of the SRX measured here correlates well with the binding of myosin to the core of the thick filament in a structure known as the interacting-head motif (or J-motif) observed previously by electron microscopy. Both the structural array and the long-lived SRX require ATP, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in skeletal or cardiac preparations. EPR spectroscopy of a spin-labeled nucleotide bound to the motor domain of myosin in relaxed tarantula fibers likewise shows orientation that is lost when the myosin is phosphorylated. Together, the data support the hypothesis that the SRX myosin and the myosin seen in the EM of the order helical array in tarantula filaments are the same.

Actin Straightens the Myosin Relay Helix during the Powerstroke

We have used transient time-resolved FRET, (TR)2FRET, to resolve the structural kinetics of the myosin relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in all myosin isoforms coupling structural changes in the ATPase site with rotation of the light chain binding domain and force generation. Previous work demonstrated that the relay helix of the myosin II motor domain from Dictyostelium Discoideum exists in dynamic equilibrium between a bent pre-powerstroke state stabilized by ATP, and a straight post-powerstroke state which predominates in the presence of ADP or when the nucleotide binding site is empty. We predict that actin binding to the myosin.ADP.Pi complex will reverse helix bending. This transition should parallel the myosin power-stroke. To test this hypothesis, a Cys-lite Dictyostelium myosin motor domain was labeled at two introduced Cys residues shown to detect relay helix bending using DEER, TR-FRET and (TR)2FRET spectroscopy. We preformed a (TR)2FRET experiment measuring the structural distribution of the relay helix every millisecond after rapid mixing of myosin.ADP/Pi with actin. This experiment showed that actin binding straightens the bent helix. We characterized the actin dependence of helix straightening and then correlated this dependence with the kinetics of actin binding and phosphate dissociation from the myosin active site. Numerical simulations were used to model a system containing pre and post-powerstroke structural states, actin binding, hydrolysis and phosphate dissociation. We globally fit this model to the (TR)2FRET, actin binding, and phosphate dissociation transients. From this analysis, we are able to determine the timing of helix straightening with respect to actin binding and the critical step of phosphate dissociation which acts as the thermodynamic driving force for myosin's force generating powerstroke.

Structural Basis for Myopathic Defects Engendered by Alterations in the Myosin Rod

While mutations in the myosin subfragment 1 motor domain can directly disrupt the generation and transmission of force along myofibrils and lead to myopathy, the mechanism whereby mutations in the myosin rod influences mechanical function is less clear. Here, we used a combination of various imaging techniques and molecular dynamics simulations to test the hypothesis that perturbations in the myosin rod can disturb normal sarcomeric uniformity and, like motor domain lesions, would influence force production and propagation. We show that disrupting the rod can alter its nanomechanical properties and, in vivo, can drive asymmetric myofilament and sarcomere formation. Our imaging results indicate that myosin rod mutations likely disturb production and/or propagation of contractile force. This provides a unifying theory where common pathological cascades accompany both myosin motor and specific rod domain mutations. Finally, we suggest that sarcomeric inhomogeneity, caused by asymmetric thick filaments, could be a useful index of myopathic dysfunction.

Mouse class III myosins: kinase activity and phosphorylation sites

As class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation.

Structural Basis for the Allosteric Interference of Myosin Function by Reactive Thiol Region Mutations G680A and G680V

The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements. Structural and functional data indicate that the mutations induce the preferential population of a state that resembles the ADP-bound state. Moreover, the Gly-680 mutants display greatly reduced dynamic properties, which appear to be related to the recovery of myosin motor function at elevated temperatures.

Two Drosophila Myosin Transducer Mutants with Distinct Cardiomyopathies Have Divergent ADP and Actin Affinities

Two Drosophila myosin II point mutations (D45 and Mhc5) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc5) could stabilize (D45) or destabilize (Mhc5) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1A261T and S1G200D) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1A261T is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin·S1), and reduced ATP-induced dissociation of actin·S1), whereas S1G200D shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin·S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1A261T (dilated) and S1G200D (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1A261T weakens the affinity of S1·ADP for actin, whereas S1G200D increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up2 mutation in Drosophila.

Loop 1 dynamics in smooth muscle myosin: isoform specific differences modulate ADP release

Isoforms of the smooth muscle (SM) myosin motor domain differ in the presence or absence of a seven amino acid insert in a flexible surface loop spanning the nucleotide-binding pocket known as Loop 1. The presence of this insert leads to a two-fold increase in actin sliding velocity and ADP release rate between these isoforms, although the effect of Loop 1 on the kinetics of ADP release remains unclear. To further investigate the role of the Loop 1 insert in modulating ADP release in SM myosin we have inserted a single tryptophan residue into Loop 1 of both isoforms as a probe of local structural dynamics. By monitoring the dynamics of Loop 1 in relation to the release of ADP we have observed a unique movement of Loop 1 in the inserted isoform, preceding nucleotide release, which is absent in the non-inserted isoform. This movement is sequence dependent as alanine replacement of the insert residues abolishes the transition and slows ADP release. Thus movement of Loop 1 is a critical factor in increasing the ADP release rate in the inserted faster isoform of SM myosin.