Myosin Motor Domain Research Articles

Allosteric modulation of cardiac myosin dynamics by omecamtiv mecarbil.

New promising avenues for the pharmacological treatment of skeletal and heart muscle diseases rely on direct sarcomeric modulators, which are molecules that can directly bind to sarcomeric proteins and either inhibit or enhance their activity. A recent breakthrough has been the discovery of the myosin activator omecamtiv mecarbil (OM), which has been shown to increase the power output of the cardiac muscle and is currently in clinical trials for the treatment of heart failure. While the overall effect of OM on the mechano-chemical cycle of myosin is to increase the fraction of myosin molecules in the sarcomere that are strongly bound to actin, the molecular basis of its action is still not completely clear. We present here a Molecular Dynamics study of the motor domain of human cardiac myosin bound to OM, where the effects of the drug on the dynamical properties of the protein are investigated for the first time with atomistic resolution. We found that OM has a double effect on myosin dynamics, inducing a) an increased coupling of the motions of the converter and lever arm subdomains to the rest of the protein and b) a rewiring of the network of dynamic correlations, which produces preferential communication pathways between the OM binding site and distant functional regions. The location of the residues responsible for these effects suggests possible strategies for the future development of improved drugs and the targeting of specific cardiomyopathy-related mutations.

Unidirectional growth of heavy meromyosin clusters along actin filaments revealed by real-time fluorescence microscopy.

Heavy meromyosin (HMM) forms clusters along actin filaments under low ATP concentrations. Here, we observed the growth of HMM clusters under low concentrations of ATP in real time using fluorescence microscopy. When actin filaments were loosely immobilized on positively charged lipid bilayers, clusters of HMM-GFP were readily formed. Time-lapse observation revealed that the clusters grew unidirectionally. When we used a mixture of actin filaments and copolymers of actin and acto-S1dC, a chimeric protein of actin and the myosin motor domain, HMM-GFP preferentially formed clusters along the copolymers. We thus suggest that binding of myosin motors carrying ADP and Pi induces unidirectional conformational changes in actin filaments and allosterically recruits more myosin binding. In contrast, when actin filaments and copolymers were anchored to glass substrate via stable biotin-avidin linkage, higher concentrations of HMM-GFP were required to form clusters than on the lipid bilayer. Moreover, actin filaments and copolymers were not discriminated regarding preferential cluster formation. This is presumably because the myosin-induced cooperative conformational changes in actin filaments involve changes in the helical twist. Consistent with this, cofilin clusters, which supertwist the helix, were readily formed along loosely immobilized actin filaments, but not along those anchored via biotin-avidin linkage.

The structure of the actin-smooth muscle myosin motor domain complex in the rigor state.

Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Å resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure.

Regulation and Plasticity of Catalysis in Enzymes: Insights from Analysis of Mechanochemical Coupling in Myosin.

The mechanism of ATP hydrolysis in the myosin motor domain is analyzed using a combination of DFTB3/CHARMM simulations and enhanced sampling techniques. The motor domain is modeled in the pre-powerstroke state, in the post-rigor state, and as a hybrid based on the post-rigor state with a closed nucleotide-binding pocket. The ATP hydrolysis activity is found to depend on the positioning of nearby water molecules, and a network of polar residues facilitates proton transfer and charge redistribution during hydrolysis. Comparison of the observed hydrolysis pathways and the corresponding free energy profiles leads to detailed models for the mechanism of ATP hydrolysis in the pre-powerstroke state and proposes factors that regulate the hydrolysis activity in different conformational states. In the pre-powerstroke state, the scissile Pγ-O3β bond breaks early in the reaction. Proton transfer from the lytic water to the γ-phosphate through active site residues is an important part of the kinetic bottleneck; several hydrolysis pathways that feature distinct proton transfer routes are found to have similar free energy barriers, suggesting a significant degree of plasticity in the hydrolysis mechanism. Comparison of hydrolysis in the pre-powerstroke state and the closed post-rigor model suggests that optimization of residues beyond the active site for electrostatic stabilization and preorganization is likely important to enzyme design.

Mutations in the SH1 helix alter the thermal properties of myosin II.

The myosin II SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain and possibly plays a key role in the arrangement of the converter/lever arm. Several point mutations within the SH1 helix in human myosin IIs have been shown to cause diseases. To reveal whether these SH1 helix mutations affect not only motile activities but also thermal properties of myosin II, here we introduced the E683K or R686C point mutation into the SH1 helix in Dictyostelium myosin II. Thermal inactivation as well as thermal aggregation rates of these mutant proteins demonstrated that these mutations decreased the thermal stability of myosin II. Temperature dependence of sliding velocities of actin filaments showed that these mutations also reduced the activation energy of a rate-limiting process involved in actin movement. Given that these mutations are likely to alter coupling between the subdomains, and thus their thermal fluctuations, we propose that the SH1 helix is a key structural element that determines the flexibility and thermal properties of the myosin motor. These characteristics of the SH1 helix may contribute to the pathogenesis of the human diseases caused by mutations within this structural element.

Altered Smooth Muscle Cell Force Generation as a Driver of Thoracic Aortic Aneurysms and Dissections.

The importance of maintaining contractile function in aortic smooth muscle cells (SMCs) is evident by the fact that heterozygous mutations in the major structural proteins or kinases controlling contraction lead to the formation of aneurysms of the ascending thoracic aorta that predispose to life-threatening aortic dissections. Force generation by SMC requires ATP-dependent cyclic interactions between filaments composed of SMC-specific isoforms of α-actin (encoded by ACTA2) and myosin heavy chain (MYH11). ACTA2 and MYH11 mutations are predicted or have been shown to disrupt this cyclic interaction predispose to thoracic aortic disease. Movement of the myosin motor domain is controlled by phosphorylation of the regulatory light chain on the myosin filament, and loss-of-function mutations in the dedicated kinase for this phosphorylation, myosin light chain kinase (MYLK) also predispose to thoracic aortic disease. Finally, a mutation in the cGMP-activated protein kinase (PRKG1) results in constitutive activation of the kinase in the absence of cGMP, thus driving SMC relaxation in part through increased dephosphorylation of the regulatory light chain and predisposes to thoracic aortic disease. Furthermore, SMCs cannot generate force without connections to the extracellular matrix through focal adhesions, and mutations in the major protein in the extracellular matrix, fibrillin-1, linking SMCs to the matrix also cause thoracic aortic disease in individuals with Marfan syndrome. Thus, disruption of the ability of the aortic SMC to generate force through the elastin-contractile units in response to pulsatile blood flow may be a primary driver for thoracic aortic aneurysms and dissections.

Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

Co-delivery of cell-wall-forming enzymes in the same vesicle for coordinated fungal cell wall formation.

Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation.

Tropomyosin isoforms bias actin track selection by vertebrate myosin Va.

Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin-Tpm3.1 and actin-Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin-Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an "actin-Tpm code" that allows for spatial and temporal sorting of actomyosin function in the cell.

An antibody against an Anopheles albimanus midgut myosin reduces Plasmodium berghei oocyst development.

BackgroundMalaria parasites are transmitted by Anopheles mosquitoes. Although several studies have identified mosquito midgut surface proteins that are putatively important for Plasmodium ookinete invasion, only a few have characterized these protein targets and demonstrated transmission-blocking activity. Molecular information about these proteins is essential for the development of transmission-blocking vaccines (TBV). The aim of the present study was to test three monoclonal antibodies (mAbs), A-140, A-78 and A-10, for their ability to recognize antigens and block oocyst infection of the midgut of Anopheles albimanus, a major malaria vector in Latin America.MethodWestern-blot of mAbs on antigens from midgut brush border membrane vesicles was used to select antibodies. Three mAbs were tested by membrane feeding assays to evaluate their potential transmission-blocking activity against Plasmodium berghei. The cognate antigens recognized by mAbs with oocyst-reducing activity were determined by immunoprecipitation followed by liquid chromatography tandem mass spectrometry.ResultsOnly one mAb, A-140, significantly reduced oocyst infection intensity. Hence, its probable protein target in the Anopheles albimanus midgut was identified and characterized. It recognized three high-molecular mass proteins from a midgut brush border microvilli vesicle preparation. Chemical deglycosylation assays confirmed the peptide nature of the epitope recognized by mAb A-140. Immunoprecipitation followed by proteomic identification with tandem mass spectrometry revealed five proteins, presumably extracted together as a complex. Of these, AALB007909 had the highest mascot score and corresponds to a protein with a myosin head motor domain, indicating that the target of mAb A-140 is probably myosin located on the microvilli of the mosquito midgut.ConclusionThese results provide support for the participation of myosin in mosquito midgut invasion by Plasmodium ookinetes. The potential inclusion of this protein in the design of new multivalent vaccine strategies for blocking Plasmodium transmission is discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1548-8) contains supplementary material, which is available to authorized users.

Detecting Structural Changes in Myosin using Bifunctional Spin Labels

Actomyosin interaction is responsible for driving a vast number of cellular processes, including force generation during muscle contraction. In this context, myosin is the molecular “motor” that produces muscle contraction. The myosin motor domain is comprised from three major subdomains: the force-generating domain (relay helix, SH-1, and converter), the ATP-binding pocket and the actin-binding cleft. During the cycle of force generation, myosin undergoes a series of structural rearrangements including “bending” of the relay helix, which communicates the biochemical state of the ATP binding pocket to the lever arm. The relay helix has previously been shown to reflect changes in distance, dynamics and orientation by electron paramagnetic resonance (EPR) with nucleotide state of the myosin catalytic domain. We now observe relay helix conformational changes using two rigidly attached bifunctional spin labels (BSL) on the relay helix (492.496) and in the lower 50 kDa domain (639.643) of the Dictyostelium myosin II motor domain by double electron electron resonance spectroscopy (DEER). We observe changes in relay helix conformation with various allosteric effectors - including potent myosin II inhibitors and physiological inhibitors such as site-directed oxidation. This approach allows for characterization of perturbations in chemo-mechanical coupling within the actomyosin complex under inhibitory conditions, as well as showing the reliability of relay helix spin probe sensitivity to the changes in myosin structural dynamics.

The Unique Roles of UNC-45 Domains in Chaperoning Myosin Folding and Modulating Myosin Powerstroke

Molecular chaperones increase de novo protein folding efficiency in the crowded environment of the cell. They typically function by shielding hydrophobic regions of unfolded and partially folded proteins, preventing misfolding and aggregation. Also, certain chaperones interact with proteins further along their folding process, including the native state, but the relevance of this role remains unclear. Investigation of the mechanisms used by the chaperones UNC-45 and Hsp90 to assist in folding the myosin motor domain offers an ideal opportunity to investigate these gaps in knowledge.The main driver of current research is our unexpected finding that the myosin chaperone UNC-45 inhibits myosin motor domain powerstroke until its partner chaperone Hsp90 reverses the inhibition. We have found that the distinct domains comprising UNC-45 participate in discretely different transactions during myosin folding and assembly: 1) the UCS domain shields misfolding of folding intermediates as they occur heat shock conditions (and hence is responsible for the ‘classic chaperone’ function); 2) Our binding studies indicate that the Central domain also interacts with myosin but does not prevent myosin aggregation thus it does not possess chaperone-like activities. We found that the Central domain alone acts as an inhibitor of myosin power stroke. This different behavior and the fact that the Central domain induces significant conformational changes within the myosin--UNC-45 complex through allosteric interactions, suggest that the Central domain performs a different function than the UCS domain; 3) the TPR domain recruits Hsp90 to allow the myosin powerstroke to resume actin translocation(‘unblocking’ function).Our studies should provide novel mechanistic insights into the pathogenesis of increased or reduced myosin activity (such as in certain hypertrophic and dilated cardiomyopathies) and its modulation by chaperones.This work was supported by the American Heart Foundation (AHA 13GRNT17290006).

High-Resolution Structural Insight into the Myosin VI-F-Actin Interface

The large superfamily of myosin motor proteins is responsible for movement and force generation at multiple scales of biology, ranging from muscle contraction to intracellular transport. While the mechanism of actomyosin has been subject to extensive biophysical, biochemical, and structural characterization, to our knowledge no high-resolution structure has been obtained of a myosin-F-actin interface, a major gap in the complete mechanistic characterization of myosins. Making use of recent methodological developments in cryo-EM, here we present a 4.5 A resolution reconstruction of an engineered myosin VI, an unconventional minus-end directed motor, in a nucleotide-free state bound to F-actin. We observe rearrangements in both the myosin motor domain and actin relative to structures of these components in isolation, suggesting a reciprocal relationship between actin and myosin conformation during force generation. In addition to providing mechanistic insights specific to myosin VI, this study paves the way for high-resolution structural analysis of diverse actomyosin complexes.

Revisiting Myosin Families Through Large-scale Sequence Searches Leads to the Discovery of New Myosins.

Myosins are actin-based motor proteins involved in many cellular movements. It is interesting to study the evolutionary patterns and the functional attributes of various types of myosins. Computational search algorithms were performed to identify putative myosin members by phylogenetic analysis, sequence motifs, and coexisting domains. This study is aimed at understanding the distribution and the likely biological functions of myosins encoded in various taxa and available eukaryotic genomes. We report here a phylogenetic analysis of around 4,064 myosin motor domains, built entirely from complete or near-complete myosin repertoires incorporating many unclassified, uncharacterized sequences and new myosin classes, with emphasis on myosins from Fungi, Haptophyta, and other Stramenopiles, Alveolates, and Rhizaria (SAR). The identification of large classes of myosins in Oomycetes, Cellular slime molds, Choanoflagellates, Pelagophytes, Eustigmatophyceae, Fonticula, Eucoccidiorida, and Apicomplexans with novel myosin motif variants that are conserved and thus presumably functional extends our knowledge of this important family of motor proteins. This work provides insights into the distribution and probable function of myosins including newly identified myosin classes.

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

Myosin isoforms and the mechanochemical cross-bridge cycle.

At the latest count the myosin family includes 35 distinct groups, all of which have the conserved myosin motor domain attached to a neck or lever arm, followed by a highly variable tail or cargo binding region. The motor domain has an ATPase activity that is activated by the presence of actin. One feature of the myosin ATPase cycle is that it involves an association/dissociation with actin for each ATP hydrolysed. The cycle has been described in detail for a large number of myosins from different classes. In each case the cycle is similar, but the balance between the different molecular events in the cycle has been altered to produce a range of very different mechanical activities. Myosin may spend most of the ATPase cycle attached to actin (high duty ratio), as in the processive myosin (e.g. myosin V) or the strain-sensing myosins (e.g. myosin 1c). In contrast, most muscle myosins spend 80% of their ATPase cycle detached from actin. Within the myosin IIs found in human muscle, there are 11 different sarcomeric myosin isoforms, two smooth muscle isoforms as well as three non-muscle isoforms. We have been exploring how the different myosin isoforms have adapted the cross-bridge cycle to generate different types of mechanical activity and how this goes wrong in inherited myopathies. The ideas are outlined here.

Identification and Clinical Implications of Novel MYO15A Mutations in a Non-consanguineous Korean Family by Targeted Exome Sequencing.

Mutations of MYO15A are generally known to cause severe to profound hearing loss throughout all frequencies. Here, we found two novel MYO15A mutations, c.3871C>T (p.L1291F) and c.5835T>G (p.Y1945X) in an affected individual carrying congenital profound sensorineural hearing loss (SNHL) through targeted resequencing of 134 known deafness genes. The variant, p.L1291F and p.Y1945X, resided in the myosin motor and IQ2 domains, respectively. The p.L1291F variant was predicted to affect the structure of the actin-binding site from three-dimensional protein modeling, thereby interfering with the correct interaction between actin and myosin. From the literature analysis, mutations in the N-terminal domain were more frequently associated with residual hearing at low frequencies than mutations in the other regions of this gene. Therefore we suggest a hypothetical genotype-phenotype correlation whereby MYO15A mutations that affect domains other than the N-terminal domain, lead to profound SNHL throughout all frequencies and mutations that affect the N-terminal domain, result in residual hearing at low frequencies. This genotype-phenotype correlation suggests that preservation of residual hearing during auditory rehabilitation like cochlear implantation should be intended for those who carry mutations in the N-terminal domain and that individuals with mutations elsewhere in MYO15A require early cochlear implantation to timely initiate speech development.

Myosin S2 Origins Track Evolution of Strong Binding on Actin by Azimuthal Rolling of Motor Domain

Myosin crystal structures have given rise to the swinging lever arm hypothesis, which predicts a large axial tilt of the lever arm domain during the actin-attached working stroke. Previous work imaging the working stroke in actively contracting, fast-frozen Lethocerus muscle confirmed the axial tilt; but strongly bound myosin heads also showed an unexpected azimuthal slew of the lever arm around the thin filament axis, which was not predicted from known crystal structures. We hypothesized that an azimuthal reorientation of the myosin motor domain on actin during the weak-binding to strong-binding transition could explain the lever arm slew provided that myosin’s α-helical coiled-coil subfragment 2 (S2) domain emerged from the thick filament backbone at a particular location. However, previous studies did not adequately resolve the S2 domain. Here we used electron tomography of rigor muscle swollen by low ionic strength to pull S2 clear of the thick filament backbone, thereby revealing the azimuth of its point of origin. The results show that the azimuth of S2 origins of those rigor myosin heads, bound to the actin target zone of actively contracting muscle, originate from a restricted region of the thick filament. This requires an azimuthal reorientation of the motor domain on actin during the weak to strong transition.

Formation of the Motor Protein-Photochromic ADP Analogue-Fluorometal Ternary Complex and Photo-Reversible Transition along the Steps in ATPase Cycle

In the presence of Mg2+ADP, myosin forms stable ternary complexes with phosphate analogues, fluoroberyllate (BeFn), fluoroaluminate (AlF4-) and orthovanadate (Vi), each of which may mimic different transient state along ATPase cycle. It is known that kiensin also forms similar ternary complexes. Our previous studies on the photoaffinity-labelling using photoactive ADP analogue and 19F NMR spectroscopy using 19F-labeled ADP analogue for the ternary complexes revealed that there is some variation in the myosin-nucleotide contacts at the nucleotide base among the ternary complexes. Previously, we have incorporated photochromic molecule, azobenzen derivative into the functional region of ATP driven motor proteins and succeeded to control activities and conformation reversibly by photochromic irradiation. Aim of this study is to induce transition between the different steps in ATPase cycle of motor proteins utilizing photoisomerization on the motor protein-phtochromic ADP analogue-fluorometal ternary complexes. In this study, three types of photochromic ATP analogues composed of azobenznene, spiropyran and fulgimide derivatives were synthesized. The photo-responsive interaction of the photochromic ATP analogues with myosin and kinesin were examined. Photo-dependent conformational changes of the myosin motor domain were monitored by X-ray small angle scattering. And the photo-reversible changes in the velocity of the microtubule glyding for kinesin in the presence of the photochromic ATP analogues were also observed. In the presence of fluorometal, BeFn, photochromic ADP analogues were trapped within the ATPase site of kinesin and myosin resulted in formation of ternary complexes. The conformational changes of the complexes induced by photo irradiation were studied.

Functional Analysis of Freeman-Sheldon Syndrome Causing Mutations on Embryonic Myosin

Freeman-Sheldon Syndrome (FSS) is a rare genetic disorder characterized primarily by multiple congenital contractures of facial muscles and distal joints, craniofacial abnormalities among other phenotypes. Mutations in embryonic skeletal myosin (MYH3) are the only known cause of FSS, and the vast majority of patients have one of three mutations: R672C, R672H, and T178I. The goal of this study was to determine the functional effects of these FSS- causing mutations on the human embryonic myosin motor domain. Preliminary data show a 10-fold reduction in ATP binding for both S1 (k2+) and actin-S1 (k′2+) for the R672C mutation. ADP affinity appears unaffected for this mutant. In the case of R672H, measurements of ATP binding were difficult because there was such a small change in tryptophan fluorescence (1.5% compared to 7-8% for Emb-WT). There was a 30 fold reduction in ATP binding to S1. Taken together with the reduction in tryptophan fluorescence change, we speculate a disruption in the conformational change in the recovery stroke. However when measured in the presence of actin we see values similar to Emb-WT. Interestingly, the affinity of ADP for actin-S1 is >3 fold tighter in this mutant with the rate constant for ADP release being reduced >3 fold. The affinity for actin is ∼20 fold weaker in the rigor complex and 10 fold tighter in the presence of ADP. For the T178I mutant which in the myosin structure forms a hydrogen bond with the R672 residue, we expect a similar effect in nucleotide binding. Our preliminary data shows a 5 fold reduction in ATP binding to S1 and 3 fold to actin-S1. When comparing to Emb-WT, the functional characteristics of these mutants are severely disrupted specifically in ATP induction of cross-bridges, which can explain the disease phenotype.