Chicken Skeletal Muscle Myosin Research Articles

Effect of low pH on single skeletal muscle myosin mechanics and kinetics

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V(actin)) in the IVM by 36%. Single molecule experiments, at 1 microM ATP, decreased the average unitary step size of myosin (d) from 10 +/- 2 nm (pH 7.4) to 2 +/- 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 microM to 1 mM at pH 6.4 restored d (9 +/- 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V(actin), however, was not restored by raising the [ATP] (1-10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t(on)). Measurement of t(on) as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t(on) by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V(actin) approximately d/t(on)), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V(actin), suggesting a molecular explanation for this component of muscular fatigue.

αB-Crystallin Maintains Skeletal Muscle Myosin Enzymatic Activity and Prevents its Aggregation under Heat-shock Stress

Here, we provide functional and direct structural evidence that alphaB-crystallin, a member of the small heat-shock protein family, suppresses thermal unfolding and aggregation of the myosin II molecular motor. Chicken skeletal muscle myosin was thermally unfolded at heat-shock temperature (43 degrees C) in the absence and in the presence of alphaB-crystallin. The ATPase activity of myosin at 25 degrees C was used as a parameter to monitor its unfolding. Myosin retained only 65% and 8% of its ATPase activity when incubated at heat-shock temperature for 15 min and 30 min, respectively. However, 84% and 58% of the myosin ATPase activity was maintained when it was incubated with alphaB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by approximately 90%, when myosin was thermally unfolded at 43 degrees C for 30 min, but was reduced by only approximately 42% when it was incubated with alphaB-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 degrees C for 30 min, while alphaB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS alphaB-crystallin fluorescence studies confirmed the transient interaction of alphaB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that approximately 94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 degrees C for 30 min. alphaB-Crystallin, however, protected approximately 48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that alphaB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, alphaB-crystallin may be critical for nascent myosin folding, promoting myofibrillogenesis, maintaining cytoskeletal integrity and sustaining muscle performance, since heat-shock temperatures can be produced during multiple stress conditions or vigorous exercise.

Loop 1 of Transducer Region in Mammalian Class I Myosin, Myo1b, Modulates Actin Affinity, ATPase Activity, and Nucleotide Access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.

Myo1c is designed for the adaptation response in the inner ear.

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADP-release mechanism, which allows it to adapt to mechanical load.

Conformation and dynamics of the SH1-SH2 helix in scallop myosin.

Atomic structures of scallop myosin subfragment 1(S1) with the bound MgADP, MgAMPPNP, and MgADP.BeF(x) provide crystallographic evidence for a destabilization of the helix containing reactive thiols SH1 (Cys703) and SH2 (Cys693). A destabilization of this helix was not observed in previous structures of S1 (from chicken skeletal, Dictyostelium discoideum, and smooth muscle myosins), including complexes for which solution experiments indicated such a destabilization. In this study, the factors that influence the SH1-SH2 helix in scallop S1 were examined using monofunctional and bifunctional thiol reagents. The rate of monofunctional labeling of scallop S1 was increased in the presence of MgADP and MgATPgammaS but was inhibited by MgADP.V(i) and actin. The resulting changes in ATPase activities of S1 were symptomatic of SH2 and not SH1 modification, which was confirmed by mass spectrometry analysis. With bifunctional reagents of various lengths, cross-linking did not occur on a short time scale in the absence of nucleotides. In the presence of MgADP, cross-linking was greatly enhanced for all of the reagents. These reactions, as well as the formation of a disulfide bond between SH1 and SH2, were much faster in scallop S1.ADP than in rabbit skeletal S1.ADP and were rate-limited by the initial attachment of the reagent to scallop S1. The cross-linking sites were mapped by mass spectrometry to SH1 and SH2. These results reveal isoform-specific differences in the conformation and dynamics of the SH1-SH2 helix, providing a possible explanation for destabilization of this helix in some scallop S1 but not in other S1 isoform structures.

Hamy3, a novel type 100 kDa myosin from sunflower.

Hamy3, a novel type myosin heavy chain from sunflower is the smallest myosin described so far, with only 900 amino acid residues. One interesting finding in Hamy3 is the glycine to glutamine alteration at residue 741, which corresponds to chicken skeletal muscle myosin glycine 699 (G699). G699 is found in 125 out of 129 myosin sequences and is interpreted in terms of its role as a pivot point for motion in the myosin "lever arm hypothesis". Changes in this crucial part of myosin might indicate a role that is different from the generation of intracellular motility.

Light-induced proteolysis of myosin heavy chain by Rose Bengal-conjugated antibody complexes

The specific light-induced, non-enzymatic digestion of chicken skeletal muscle myosin heavy chain by xanthene dye-conjugated antibodies is reported. The xanthene dye Rose Bengal was conjugated to either a mouse monoclonal anti-myosin primary specific antibody or to goat anti-mouse IgG secondary antibodies. Under our experimental conditions, visible light induced the non-enzymatic breakdown of myosin heavy chains when chicken skeletal muscle myosin either directly formed a complex with Rose Bengal-conjugated anti-myosin antibodies or indirectly formed a complex with anti-myosin antibody-Rose Bengal-conjugated secondary antibodies. The rate of the photochemical reaction depended on irradiation time and temperature. Although SDS–PAGE and immunoblot analyses showed that fragments migrating below the myosin heavy chain polypeptide predominated, these analyses also showed higher molecular mass polypeptides were generated.

Actin Residue Glu93 Is Identified as an Amino Acid Affecting Myosin Binding

Many mutants have been described that affect the function of the actin encoded by the Drosophila melanogaster indirect flight muscle-specific actin gene, Act88F. We describe the development of procedures for purification of this actin from the other isoforms expressed in the fly as well as in vitro motility, single molecule force/displacement measurements, and stop-flow solution kinetic studies of the wild-type actin and that of the E93K mutation of the Act88F gene. We show that this mutation affects in vitro motility of F-actin, in both the presence and absence of methylcellulose, and the ability of the ACT88F actin to bind the S1 fragment of rabbit skeletal myosin. However, optical tweezer measurements of the actomyosin working stroke and the force transmitted from the rabbit heavy meromyosin to and through F-actin are unchanged by the mutation. These results support the proposal (Holmes, K. C. (1995) Biophys J. 68, (suppl.) 2-7) that actin residue Glu(93) is part of the secondary myosin binding site and suggest that myosin binding occurs first at the primary myosin binding site and then at the secondary site.

Predicting allosteric switches in myosins.

The sequences of several members of the myosin family of molecular motors are evaluated using ASP (Ambivalent Structure Predictor), a new computational method. ASP predicts structurally ambivalent sequence elements by analyzing the output from a secondary structure prediction algorithm. These ambivalent sequence elements form secondary structures that are hypothesized to function as switches by undergoing conformational rearrangement. For chicken skeletal muscle myosin, 13 discrete structurally ambivalent sequence elements are identified. All 13 are located in the heavy chain motor domain. When these sequence elements are mapped into the myosin tertiary structure, they form two compact regions that connect the actin binding site to the adenosine 5'-triphosphate (ATP) site, and the ATP site to the fulcrum site for the force-producing bending of the motor domain. These regions, predicted by the new algorithm to undergo conformational rearrangements, include the published known and putative switches of the myosin motor domain, and they form plausible allosteric connections between the three main functional sites of myosin. The sequences of several other members of the myosin I and II families are also analyzed.

A Long, Weakly Charged Actin-binding Loop Is Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin

Chimeric substitution of the weak actin-binding loop (ABL) from chicken skeletal muscle myosin for that of gizzard smooth muscle heavy meromyosin (HMM) causes activation of the dephosphorylated mutant (SABL HMM; Rovner, A. S., Freyzon, Y., and Trybus, K. M. (1995) J. Biol. Chem. 270, 30260-30263). The present study determined whether this loss of regulation is due to the greater positive charge density (5 versus 3 clustered lysine residues) or lesser length (14 versus 26 residues) of the mutant ABL. Charge augmentation had little effect on regulation of expressed mutants, but elimination of the 12 N-terminal amino acids from the wild-type ABL significantly increased actin-activated ATPase activity of the dephosphorylated relative to the phosphorylated molecule while conferring the ability to move actin filaments in vitro on the former. Addition of the same 12 residues to the SABL mutant increased the ratio of phosphorylated to dephosphorylated ATPase activity while imparting wild type-like regulation to motility. However, full actin activation of dephosphorylated ATPase activity required both the shorter length and greater positive charge density found in the SABL loop. These results demonstrate that, compared with skeletal, both the greater length and lesser positive charge density of the smooth muscle myosin ABL are required for proper phosphorylation-mediated regulation of the molecule.

Subunit Interactions within an Expressed Regulatory Domain of Chicken Skeletal Myosin: LOCATION OF THE NH2 TERMINUS OF THE REGULATORY LIGHT CHAIN BY FLUORESCENCE RESONANCE ENERGY TRANSFER

The regulatory domain (RD), or neck region of the myosin head, consists of two classes of light chains that stabilize an alpha-helical segment of the heavy chain. RD from chicken skeletal muscle myosin was prepared in Escherichia coli by coexpression of a 9-kDa heavy chain fragment with the essential light chain. Recombinant regulatory light chain (RLC), wild type or mutant, was added separately to reconstitute the complex. The affinity of RD for divalent cations was determined by measuring the change in fluorescence of a pair of heavy chain tryptophans upon addition of calcium or magnesium. The complex bound divalent cations with high affinity, similar to the association constants determined for native myosin. The intrinsic fluorescence of the tryptophans could be used as a donor to measure the fluorescence resonance energy transfer distance to a single labeled cysteine engineered at position 2 on RLC. Dansylated Cys2 could also serve as a donor by preparing RLC with a second cysteine at position 79 which was labeled with an acceptor probe. These fluorescence resonance energy transfer distances (24-30 A), together with a previous measurement between Cys2 and Cys155 (Wolff-Long, V. L., Tao, T., and Lowey, S. (1995) J. Biol. Chem. 270, 31111-31118) suggest a location for the NH2 terminus of RLC that appears to preclude a direct interaction between the phosphorylatable serine and specific residues in the COOH-terminal domain.

Brush border myosin-I structure and ADP-dependent conformational changes revealed by cryoelectron microscopy and image analysis.

Brush border myosin-I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751-753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain-binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The approximately 31 degrees swing corresponds to approximately 63 A at the end of the rigid light chain-binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.

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.

A myosin family reunion.

The cover photo of this issue and Fig. 1 depicts a phylogenetic tree of the ever-expanding myosin superfamily, which groups myosins into 12 different classes. For simplicity only selected members of some of the more populated classes are shown. More detailed phylogenetic analyses of the myosin I and myosin II family are shown in Fig. 2. To date, the best represented classes remain those of the ‘conventional myosin 11s and the now well studied myosin Is. There are multiple members of these two families present within a given species. For example, at least 11 myosin II genes exist in a given mammalian organism. In addition, it is clear that there are at least four myosin I genes, two genes each for myosins V, VII, and IX, and a gene for myosin X. Even in simple unicellular organisms there is diversity within a given class. Dictyostelium has at least five myosin I genes (see Fig. 2A). The phylogenetic analyses suggest that many (possibly most) of the classes will be ubiquitous in eukaryotes (Mooseker & Cheney, 1995; Sellers & Goodson, 1995). Many questions can be raised about phylogenetic analyses and the general usefulness of this approach will be discussed later. One question concerns the definition of myosin. The simplest definition is to include all molecules with a marked sequence homology to the head of conventional myosins. All of the proteins shown in the tree have a wellconserved myosin motor domain that can be aligned in primary sequence with that of chicken skeletal muscle myosin (defined as residues 89-709 of this myosin; Sellers & Goodson, 1995). In some cases there are deletions or insertions compared to the sequence of chicken skeletal muscle myosin. How-

Structure of the regulatory domain of scallop myosin at 2 A resolution: implications for regulation.

In contrast to the myosins of vertebrate skeletal muscle, molluscan myosins are regulated molecules whose enzymatic activity is switched on by the direct binding of Ca2+. The head portion (S1) of the molecule consists of a motor domain and a regulatory domain (RD) containing a 'regulatory' and an 'essential' light chain (RLC and ELC, respectively). The structures of scallop myosin RD with bound Ca2+, as well as the S1 fragment of chicken skeletal muscle myosin, have been determined previously to 2.8 A resolution. We have determined the structure at 2.0 A resolution of scallop myosin RD with bound Ca2+. The unusual coordination at the specific Ca(2+)-binding site in the ELC has now been clarified, as has the structural basis for Mg2+ binding to the RLC. A comparison of the scallop RD structure with that in the chicken S1 structure shows differences in the bending of the two RDs in two different places. Based on these structural results, a model for regulation is proposed in which the Ca(2+)-bound RD is a rigid structure, and transient flexibility of the Ca(2+)-free RD allows the myosin heads to make stabilizing intramolecular linkage which shut off the motor.

The radius of gyration of native and reductively methylated myosin subfragment-1 from neutron scattering

Reductive methylation of nearly all lysine groups of myosin subfragment-1 (S1) was required for crystallization and solution of its structure at atomic resolution. Possible effects of such methylation on the radius of gyration of chicken skeletal muscle myosin S1 have been investigated by using small-angle neutron scattering. In addition, we have investigated the effect of MgADP.Vi, which is thought to produce an analog of the S1.ADP.Pi state, on the S1 radius of gyration. We find that although methylation of S1, with or without SO42- ion addition, does not significantly alter the structure, addition of ADP plus vanadate does decrease the radius of gyration significantly. The S1 crystal structure predicts a radius of gyration close to that measured here by neutron scattering. These results suggest that the overall shape by crystallography resembles nucleotide-free S1 in solution. In order to estimate the effect of residues missing from the crystal structure, the structure of missing loops was estimated by secondary-structure prediction methods. Calculations using the complete crystal structure show that a simple closure of the nucleotide cleft by a rigid-body torsional rotation of residues (172-180 to 670) around an axis running along the base of the cleft alone does not produce changes as large as seen here and in x-ray scattering results. On the other hand, a rigid body rotation of either the light-chain binding domain (767 to 843 plus light chains) or of a portion of 20-kDa peptide plus this domain (706 to 843 plus light chains) is more readily capable of producing such changes.

X-ray Structures of the Myosin Motor Domain of Dictyostelium discoideum Complexed with MgADP.cntdot.BeFx and MgADP.cntdot.AlF4-

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-MgADP complex belong to space group P2(1)2(1)2 with unit cell parameters of a = 105.3 A, b = 182.6 A, and c = 54.7 A, whereas the crystals of the aluminum fluoride complex belong to the orthorhombic space group C222(1) with unit cell dimensions of a = 87.9 A, b = 149.0 A, and c = 153.8 A. Chemical modification was not necessary to obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADP.BeFx is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADP.AlF4- exhibits significant domain movements. The structures suggest that the MgADP.BeFx complex mimics the ATP bound state and the MgADP.AlF4- complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADP.AlF4- complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain.

Structural interpretation of the mutations in the beta-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy.

In 10-30% of hypertrophic cardiomyopathy kindreds, the disease is caused by > 29 missense mutations in the cardiac beta-myosin heavy chain (MYH7) gene. The amino acid sequence similarity between chicken skeletal muscle and human beta-cardiac myosin and the three-dimensional structure of the chicken skeletal muscle myosin head have provided the opportunity to examine the structural consequences of these naturally occurring mutations in human beta-cardiac myosin. This study demonstrates that the mutations are related to distinct structural and functional domains. Twenty-four are clustered around four specific locations in the myosin head that are (i) associated with the actin binding interface, (ii) around the nucleotide binding site, (iii) adjacent to the region that connects the two reactive cysteine residues, and (iv) in close proximity to the interface of the heavy chain with the essential light chain. The remaining five mutations are in the myosin rod. The locations of these mutations provide insight into the way they impair the functioning of this molecular motor and also into the mechanism of energy transduction.

Myosin structure/function: a combined mutagenesis-crystallography approach

In the past year, the structure of the regulatory domain of scallop myosin has joined that of the chicken skeletal muscle myosin subfragment 1 and provided insights into the regulation of myosin function. Mutagenesis studies in a variety of systems have used the information provided by these structures to create mutant myosins to test models of chemomechanical transduction and its regulation.

Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function.

Regulatory light chains, located on the 'motor' head domains of myosin, belong to the family of Ca2+ binding proteins that consist of four 'EF-hand' subdomains. Vertebrate regulatory light chains can be divided into two functional classes: (i) in smooth/non-muscle myosins, phosphorylation of the light chains by a calcium/calmodulin-dependent kinase regulates both interaction of the myosin head with actin and assembly of the myosin into filaments, (ii) the light chains of skeletal muscle myosins are similarly phosphorylated, but they play no apparent role in regulation. To discover the basis for the difference in regulatory properties of these two classes of light chains, we have synthesized in Escherichia coli, chimeric mutants composed of subdomains derived from the regulatory light chains of chicken skeletal and smooth muscle myosins. The regulatory capability of these mutants was analysed by their ability to regulate molluscan myosin. Using this test system, we identified the third subdomain of the regulatory light chain as being responsible for controlling not only the actin-myosin interaction, but also myosin filament assembly.