Region Of Myosin Research Articles

THERMAL STABILITY OF SYNTHETIC PEPTIDES MIMICKING THE SEQUENCE OF THE REGION CONTAINING THE SKIP RESIDUES IN SQUID MYOSIN ROD

Myosin is the major protein in skeletal muscles including those of fish and shellfish. The characteristics of this protein are closely related to the biological function and the quality and physical properties of muscle<br />food. In the myosin rod (the coiled-coil region of myosin), several amino acid residues, known as skip residues, seem to destabilize the ordered structure (heptad repeat). These residues might be responsible for reducing thermal stability. Attempts were thus made to examine the role of these residues in the rod of squid myosin, based on the thermodynamic properties of synthetic peptides which have been designed to mimic the partial sequence of myosin heavy chain from the squid Todarodes pacificus mantle muscle. Five peptides, namely, with the sequence of Trp1343 -Ala1372 having the skip residue Glu1357 at the center (Peptide WT), without the skip residue (Peptide Δ), with the replacements of the skip residue (Glu) by Ile, Gln and Pro (Peptides E/I, E/Q, and E/P, respectively) to modify the helix forming propensity, were synthesized. The results obtained showed that the stability of the peptides as measured by circular dichroism spectrometry was in the order of Peptide Δ > Peptide WT > Peptide E/Q > Peptide E/P > Peptide E/I. It is suggested that the presence of the skip residues dexterously tunes the stability or flexibility of the coiled-coil structure, thus possibly regulating thick filament formation and further gel formation ability of myosin.

Biophysical Analysis of the Putative Heart Failure Drug Omecamtiv Mecarbil

The small molecule omecamtiv mecarbil (OM), currently in late phase-2 clinical trials for treatment of heart failure, activates force generation by cardiac myosin. How OM increases force generation is not known. Crosslinking and molecular docking studies suggest that the compound binds to the relay/SH1/converter region of myosin, hypothesized to control the actin-activated pre to post power-stroke structural transition in the myosin catalytic and light-chain domains. These transitions drive force production by myosin. We are currently using time-resolved FRET and EPR distance measurements to detect the pre- and post-power-stroke structural states of myosin. We have performed these studies on tissue-purified β-cardiac and α-skeletal myosin, and on a recombinant cys-lite myosin II catalytic domain model from Dictyostelium discoideum. In each of the myosin isoforms, we find that OM binding increases the mole fraction of the myosin post power-stroke structural state in the presence of ATP analogs. Transient time-resolved FRET experiments, mixing FRET-labeled myosin with ATP, show that drug binding also alters the structural kinetics of the myosin recovery-stroke. Ongoing studies are investigating the affect of OM on the actin-activated power-stroke. These results show that OM has a direct effect on the power-stroke structural transition that drives force generation in muscle, thus providing structural insight into the mechanism of this new potential therapy for heart failure. Our results also highlight the utility of structure-based time-resolved FRET and EPR assays for the discovery and characterization of allosteric enzyme modulators.

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.

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.

Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399-4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a K(a) of (2.44 +/- 0.09) x 10(5) m(-1) with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a K(a) of (5.83 +/- 0.05) x 10(4) m(-1) with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca(2+) sensitivity in that in the presence of Ca(2+) complex formation was reduced.

Action of Ascorbic Acid on a Myosin Molecule Derived from Carp

The influence of L-ascorbic acid at 40 degrees C incubation on the subfragment-1 and rod regions, prepared by chymotryptic digestion of myosin, and myosin was investigated by SDS-polyacrylamide gel electrophoresis and transmission electron microscopy respectively. It was observed that L-ascorbic acid acted more readily on the subfragment-1 region of myosin. Further, circular dichroism measurement indicated that L-ascorbic acid did not affect the structure of myosin. These results suggest that L-ascorbic acid acts more readily on the myosin subfragment-1 region and promotes the gelation of myosin without producing a conformational change in this protein.

The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.

Evidence for the involvement of myosin subfragment 2 in muscle contraction.

Muscle contraction results from alternate formation and breading of cross-links between the myosin head (subfragment 1; S-l), extending from the thick filament and a neighboring thin filament (Huxley A. F., 1957; Huxley H. E., 1969). The energy for contraction is supplied by ATP hydrolysis. Since the ATPase activity and actin binding site are localized in the S-l region of myosin, S-l is commonly believed to play a major role in muscle contraction. In fact, in vitro motility assay experiments have shown that S-l alone is sufficient to produce force and move actin filaments. However, the ATP-dependent actin-myosin sliding observed in the assay systems is not the same as that actually taking place in muscle.

Physicochemical changes and mechanism of heat-induced gelation of arrowtooth flounder myosin.

Physicochemical changes of myosin during heating were investigated to elucidate the mechanism of heat-induced gelation of arrowtooth flounder (ATF) myosin at high ionic strength. Changes in dynamic properties indicated ATF myosin formed a gel in three different stages as shown by the first increase in G' (storage modulus) at 28 degrees C, followed by the decrease at 35 degrees C and the second increase at 42 degrees C. DSC thermogram showed the onset of myosin denaturation at 25 degrees C with two maximum transition temperatures at 30 and 36 degrees C. The decrease in alpha-helical content indicated ATF myosin began to unfold at 15 degrees C and the unfolding continued until it reached 65 degrees C. Turbidity measurement showed myosin began to aggregate at 23 degrees C and the aggregation was complete at 40 degrees C. Surface hydrophobicity increased consistently in the temperature range studied, 20-65 degrees C. Sulfhydryl contents decreased significantly at 20-30 degrees C due to the formation of disulfide linkages but remained constant at temperatures >30 degrees C. ATF myosin was shown to be extremely sensitive to heat, resulting in denaturation at lower temperature than other fish myosin. Denaturation was initiated by unfolding of the alpha-helical region in myosin followed by exposure of hydrophobic and sulfhydryl residues, which are subsequently involved in aggregation and gelation processes.

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.

Molecular characterization of myosin IB from the lower eukaryote Entamoeba histolytica, a human parasite

The complete amino acid sequence of the Entamoeba histolytica unconventional myosin IB (Eh-myosin IB) is reported. Sequencing of overlapping cDNA fragments reveals a single open reading frame which predicts a 130 kDa protein of 1049 aa. Eh-myosin IB presents the three characteristics domains of myosins I subclass 1. This protein presents homology with myosins IB from other amoebae, but striking homologies with vertebrate unconventional myosins were also observed. The predicted actin and ATP-binding sites are located in the head domain. The heavy chain phosphorylation region is homologous to metazoan myosins I with an acidic residue present at the phosphorylation site. In the neck domain, an IQ motif indicates potential binding of calmodulin to the myosin I heavy chain. In the tail of Eh-myosin IB the three characteristic regions of myosin I are found. A putative membrane binding domain a very short domain rich in alanine and proline we demonstrate to be functional for actin binding, and the src-homology 3 domain. The Entamoeba histolytica myosin IB is the first unconventional myosin so far described in a lower eukaryote.

Myogenic cells express multiple myosin isoforms.

In vivo and in vitro, proliferating motile myoblasts form aligned groups of cells, with a characteristic bipolar morphology, subsequently become post-mitotic, begin to express skeletal myosin and fuse. We were interested in whether members of the myosin superfamily were involved in myogenesis. We found that the myoblasts expressed multiple myosin isoforms, from at least five different classes of the myosin superfamily (classes I, II, V, VII and IX), using RT-PCR and degenerate primers to conserved regions of myosin. All of these myosin isoforms were expressed most highly in myoblasts and their expression decreased as they differentiated into mature myotubes, by RNAse protection assays, and Western analysis. However, only myosin I alpha, non-muscle myosin IIA and IIB together with actin relocalize in response to the differentiative state of the cell. In single cells, myosin I alpha was found at the leading edge, in rear microspikes and had a punctate cytoplasmic staining, and non-muscle myosin was associated with actin bundles as previously described for fibroblasts. In aligned groups of cells, all these proteins were found at the plasma membrane. Co-staining for skeletal myosin II, and myosin I alpha showed that myosin I alpha also appeared to be expressed at higher levels in post-mitotic myoblasts that had begun to express skeletal myosin prior to fusion. In early myotubes, actin and non-muscle myosin IIA and IIB remained localized at the membrane. All of the other myosin isoforms we looked at, myosin V, myosin IX and a second isoform of myosin I (mouse homologue to myr2) showed a punctate cytoplasmic staining which did not change as the myoblasts differentiated. In conclusion, although we found that myoblasts express many different isoforms of the myosin superfamily, only myosin I alpha, non-muscle myosin IIA and IIB appear to play any direct role in myogenesis.

Chicken Skin Composition As Affected By Aqueous Washing

ABSTRACTBroiler skin was washed in either 0.1M sodium chloride (pH 7.0) or 0.5% sodium bicarbonate buffer (pH 8.4) to reduce water‐soluble proteins and fat. Washing increased moisture and total protein content (P<0.05) of skin. Ash content was lower (P<0.05) in skin washed in 0.5% sodium bicarbonate buffer compared to unwashed and 0.1M sodium chloride washed skin. Fat content decreased (P<0.05) following washing. Salt extractable proteins and collagen content remaining in skin increased following washing (P<0.05). Water‐soluble proteins remaining in washed skin were lower (P<0.05). SDS‐PAGE gel patterns for salt extractable proteins remaining in skin after washing revealed more intense bands in regions of myosin (200K daltons), M‐proteins (165K daltons) and C‐protein (135–140K daltons) and 31‐45K daltons.

Distance measurements near the myosin head-rod junction using fluorescence spectroscopy

We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region.

Inhibition of myosin ATPase activity by human myasthenia gravis antibodies reactive with the acetylcholine receptor

Antibodies, obtained from myasthenia gravis patients, with reactivity for an immunodominant region of the nieotinic acetylcholine receptor were found to also react with muscle myosin. Since amino acid sequence analyses have previously suggested possible serological relationships between AChR and a head region sequence of myosin heavy chain, cross-reactive antibodies were examined for their ability to interfere with ATPase activities associated with this region of myosin. Results indicated that AChR-specific antibodies purified from MG patient serum by binding to and elution from antigen columns were found to inhibit Ca 2+-dependent, myosin-associated ATPase activity; interestingly, this inhibition appeared to be relatively selective in that neither K +(EDTA)-dependent nor Mg 2+-dependent ATPase activities were sensitive to antibody-mediated interference.

Predicting Coiled Coils from Protein Sequences

The probability that a residue in a protein is part of a coiled-coil structure was assessed by comparison of its flanking sequences with sequences of known coiled-coil proteins. This method was used to delineate coiled-coil domains in otherwise globular proteins, such as the leucine zipper domains in transcriptional regulators, and to predict regions of discontinuity within coiled-coil structures, such as the hinge region in myosin. More than 200 proteins that probably have coiled-coil domains were identified in GenBank, including alpha- and beta-tubulins, flagellins, G protein beta subunits, some bacterial transfer RNA synthetases, and members of the heat shock protein (Hsp70) family.

Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP.

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5-nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATP, the cross-bridges were also disordered. We conclude (a) that Ca2+ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca2+.

Lack of communication between LC2 light chain and the SH 1 region of myosin S-1 studied by 19F-NMR

Myosin subfragment-1 (S-1) which contains the LC2 light chain has been labelled with fluorine to allow an 19F-NMR study of the coupling and energetics of structural changes in the myosin head. Two fluorine-containing reagents, N-4-(trifluoromethyl)phenyl iodoacetamide and N-3,5-di(trifluoromethyl)phenyl iodoacetamide, have been used to label the myosin heavy chain at the unusually reactive sulfhydryl-1 (SH 1) position. The chemical shift of both reagents on S-1 is sensitive to a structural transition in the region of SH 1 which occurs upon increasing the temperature from 0°C to 35°C. The midpoint of the transition in both papain and chymotryptic S-1 is at approximately 11°C at pH 7 (0.1 M CKI). The temperature dependence of the chemical shift may be fit assuming a two-state equilibrium where ΔG°′( T) = 101–110 T + 0.386 T 2 (where T is the temperature in Kelvin). Both ΔH°′( T) and ΔS°′( T) have a small temperature dependence from 0 to 35°C: at 20°C, ΔH°′( T) = −33 kcal/mol. ΔS°′( T) = −116 e.u. and ΔC P = −226 cal/mol per deg (pH 7.0, 0.1 M KCl). The NMR data indicate that the presence of the LC2 light chain in papain S-1 does not modify the structure of S-1 in the vicinity of SH 1, nor does it modify the energetics of the structural transition from that seen in its absence with chymotryptic S-1. The presence of calcium which is bound by the LC2 of papain S-1 also does not alter the energetics of the transition. Thus it would appear that the LC2 light chain (on myosin S-1) does not participate in the two-state transition, nor does it interact strongly with regions of the heavy chain which participate in the transition.

Conformational changes in the myosin subfragment-2: Effect of pH on synthetic rod filaments

Chemical cross-linking and chymotryptic digestions of synthetic rod filaments were carried out in order to examine the conformational properties of the subfragment-2 (S-2) element. In these experiments, molded after those of K. Sutoh, Y. C. Chiao and W. F. Harrington, the rates of S-2 and light meromyosin (LMM) cross-linking were followed over the pH range from 7.0 to 8.3. The normalized rate of S-2 crosslinking, k s − 2 k LMM , decreased sharply in this pH range, in analogy with previous observations on myosin filaments, myofibrils, and glycerinated fibers. The proteolytic susceptibility of rod filaments greatly increased over the same pH range, mimicking a similar increase that has been observed in myosin filaments. These results demonstrate that the S-2 region of myosin is readily affected by small changes in charge balance and suggest that conformational changes in this part of the molecule may lead to changes in the disposition of myosin heads.