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Charge interactions between alpha-helical coiled-coil proteins have been postulated to determine the alignment of many filamentous proteins, such as myosin heavy-chain rod, paramyosin and alpha-keratin. Here we determined the sequence changes in nine mutations in the unc-15 paramyosin gene of Caenorhabditis elegans, including one nonsense, four missense, one deletion and three suppressor mutations. These mutation sites were located on a molecular model, constructed by optimizing charge interactions between paramyosin rods. Remarkably, single charge reversals (e.g., glutamic acid to lysine) were found that either disrupted or restored filament assembly in vivo. The positions of the mutations within the paramyosin molecule support the models of paramyosin assembly and further suggest that the C-terminal region containing a cluster of five mutations, and a site interacting with it, play a key role in assembly. One amino acid substitution in this C-terminal region, in which there is a "weak spot", led to a loss of reactivity with one monoclonal anti-paramyosin antibody. The results demonstrate how a single amino acid substitution can alter the assembly properties of alpha-helical molecules.
J Mol Biol 1991 Jun 05
PMID:Single charge change on the helical surface of the paramyosin rod dramatically disrupts thick filament assembly in Caenorhabditis elegans. 205 82

Chemically skinned, relaxed bundles of fibers from the striated adductor muscle of the scallop Placopecten magellanicus were rapidly frozen and freeze-substituted. In the electron microscope, ultrathin transverse sections of embedded specimens showed, in many cases, clear regularly organized projections (crossbridges) protruding from the backbones of the myosin filaments. In the majority of cases the number of projections was directly observed to be seven: this was confirmed by alignment and averaging of the images using correlation methods. The rotational power spectrum of the average image showed a strong peak at N = 7. Tilting of sections in the electron microscope showed that the long-pitch crossbridge helices were right-handed. These and other observations confirm directly the essential features of the low-resolution three-dimensional helical reconstruction of negatively stained scallop filaments calculated previously.
J Mol Biol 1991 Jul 05
PMID:Direct determination of myosin filament symmetry in scallop striated adductor muscle by rapid freezing and freeze substitution. 206 11

The established observations and unresolved questions in the assembly of myosin are outlined in this article. Much of the background information has been obtained in classical experiments using the myosin and thick filaments from vertebrate skeletal muscle. Current research is concerned with problems of myosin assembly and structure in smooth muscle, a broad spectrum of invertebrate muscles, and eukaryotic cells in general. Many of the general questions concerning myosin assembly have been addressed by a combination of genetic, molecular, and structural approaches in the nematode Caenorhabditis elegans. Detailed analysis of multiple myosin isoforms has been a prominent aspect of the nematode work. The molecular cloning and determination of the complete sequences of the genes encoding the four isoforms of myosin heavy chain and of the myosin-associated protein paramyosin have been a major landmark. The sequences have permitted a theoretical analysis of myosin rod structure and the interactions of myosin in thick filaments. The development of specific monoclonal antibodies to the individual myosins has led to the delineation of the different locations of the myosins and to their special roles in thick filament structure and assembly. In nematode body-wall muscles, two isoforms, myosins A and B, are located in different regions of each thick filament. Myosin A is located in the central biopolar zones, whereas myosin B is restricted to the flanking polar regions. This specific localization directly implies differential behavior of the two myosins during assembly. Genetic and structural experiments demonstrate that paramyosin and the levels of expression of the two forms are required for the differential assembly. Additional genetic experiments indicate that several other gene products are involved in the assembly of myosin. Structural studies of mutants have uncovered two new structures. A core structure separate from myosin and paramyosin appears to be an integral part of thick filaments. Multifilament assemblages exhibit multiple nascent thick filament-like structures extending from central paramyosin regions. Dominant mutants of myosin that disrupt thick filament assembly are located in the ATP and actin binding sites of the heavy chain. A model for a cycle of reactions in the assembly of myosin into thick filaments is presented. Specific reactions of the two myosin isoforms, paramyosin, and core proteins with multifilament assemblages as possible intermediates in assembly are proposed.
Mol Neurobiol
PMID:Genetic analysis of myosin assembly in Caenorhabditis elegans. 207 18

We have previously shown that actin from Tetrahymena pyriformis has a very divergent primary structure (Hirono, M., Endoh, H., Okada, N., Numata, O., & Watanabe, Y. (1987) J. Mol. Biol. 194, 181-192) and that though it shares essential properties with skeletal muscle actin, it does not interact at all with phalloidin or DNase I (Hirono, M., Kumagai, Y., Numata, O., & Watanabe, Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79). In this study, we investigated the copolymerization of this actin with skeletal muscle actin by direct observation of the heteropolymers formed from the two actins by means of electron microscopy. We also examined the binding of actin-binding proteins from skeletal muscle or smooth muscle to Tetrahymena actin by means of a cosedimentation assay. The results show that (i) Tetrahymena actin copolymerizes with skeletal muscle actin and that (ii) muscle myosin subfragment 1 binds to it in the absence of ATP, like skeletal muscle actin. However, it was also shown that (iii) muscle alpha-actinin hardly binds to Tetrahymena actin and that (iv) muscle tropomyosin does not bind to it at all. The results show that Tetrahymena actin has both properties similar and dissimilar to those of skeletal muscle actin.
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PMID:Tetrahymena actin: copolymerization with skeletal muscle actin and interactions with muscle actin-binding proteins. 211 Jan 55

The relationships among 153 EF-hand (calcium-modulated) proteins of known amino acid sequence were determined using the method of maximum parsimony. These proteins can be ordered into 12 distinct subfamilies--calmodulin, troponin C, essential light chain of myosin, regulatory light chain, sarcoplasmic calcium binding protein, calpain, aequorin, Stronglyocentrotus purpuratus ectodermal protein, calbindin 28 kd, parvalbumin, alpha-actinin, and S100/intestinal calcium-binding protein. Eight individual proteins--calcineurin B from Bos, troponin C from Astacus, calcium vector protein from Branchiostoma, caltractin from Chlamydomonas, cell-division-cycle 31 gene product from Saccharomyces, 10-kd calcium-binding protein from Tetrahymena, LPS1 eight-domain protein from Lytechinus, and calcium-binding protein from Streptomyces--are tentatively identified as unique; that is, each may be the sole representative of another subfamily. We present dendrograms showing the relationships among the subfamilies and uniques as well as dendrograms showing relationships within each subfamily. The EF-hand proteins have been characterized from a broad range of organismal sources, and they have an enormous range of function. This is reflected in the complexity of the dendrograms. At this time we urge caution in assigning a simple scheme of gene duplications to account for the evolution of the 600 EF-hand domains of known sequence.
J Mol Evol 1990 Jun
PMID:Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. 211 31

The cardiac changes resulting from mechanical overload of the left ventricle have been well documented and a variety of compensatory mechanisms described. These include a decrease in maximum velocity (V0) of shortening in the absence of reduction in active tension (P0), and a reversible decrease in myofibrillar adenosine triphosphatase activity resulting from isoenzymic shift from, predominantly, a form of myosin with high ATPase activity (V1) to another with low (V3). The thermodynamic advantage of the transition is the hypertrophied muscle possesses a more energy-efficient form of contraction. These reversible transitions resulted from altered gene expression of isoenzymic forms of myosin heavy chain. It must be borne in mind that the adaptational modifications just described appear to occur only in smaller animals such as the rat, that possesses several myosin isozymes. In large mammals it is mainly the V3 form of myosin that is present, which does not change with altered contractile state. Responses of the large arteries to hypertension have been poorly studied. This is surprising when one recalls that degenerative disease of such vessels, that include the aorta, carotids and ileo-femoral arteries is almost an obligatory concomitant of hypertension. Such studies as have been carried out indicate that hyperplasia is specific for abdominal aortic stenosis while hypertrophy is found in aortic smooth muscle in rats with systemic hypertension. Mechanically, an increase in V0 with no change in P0 have been reported; an increase in myofibrillar ATPase activity was also reported. Though two myosin heavy chain isozymes have been found in aortic smooth muscle densitometry did not reveal any difference in distribution between tissues from control and hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1990 Mar 05
PMID:Cardiovascular adaptations to mechanical overload. 213 92

We have estimated the step size of the myosin cross-bridge (d, displacement of an actin filament per one ATP hydrolysis) in an in vitro motility assay system by measuring the velocity of slowly moving actin filaments over low densities of heavy meromyosin on a nitrocellulose surface. In previous studies, only filaments greater than a minimum length were observed to undergo continuous sliding movement. These filaments moved at the maximum speed (Vo), while shorter filaments dissociated from the surface. We have now modified the assay system by including 0.8% methylcellulose in the ATP solution. Under these conditions, filaments shorter than the previous minimum length move, but significantly slower than Vo, as they are propelled by a limited number of myosin heads. These data are consistent with a model that predicts that the sliding velocity (v) of slowly moving filaments is determined by the product of vo and the fraction of time when at least one myosin head is propelling the filament, that is, v = vo [1-(1-ts/tc)N], where ts is the time the head is strongly bound to actin, tc is the cycle time of ATP hydrolysis, and N is the average number of myosin heads that can interact with the filament. Using this equation, the optimum value of ts/tc to fit the measured relationship between v and N was calculated to be 0.050. Assuming d = vots, the step size was then calculated to be between 10nm and 28 nm per ATP hydrolyzed, the latter value representing the upper limit. This range is within that of geometric constraint for conformational change imposed by the size of the myosin head, and therefore is not inconsistent with the swinging cross-bridge model tightly coupled with ATP hydrolysis.
J Mol Biol 1990 Aug 05
PMID:Myosin step size. Estimation from slow sliding movement of actin over low densities of heavy meromyosin. 214 85

The cardiac effects of excess growth hormone (GH) were studied in the intact adult rat and in tissues prepared from the rat. Female Wistar-Furth rats were inoculated with a clonal cell line of pituitary cells which secrete GH. Five weeks later, heart weight had increased 37% compared to control (P less than 0.01) due to concomitant increases in left and right ventricular weight. Hemodynamic measurements in the anesthetized rat showed that GH stimulated rats had a decrease in blood pressure and heart rate and a small increase of left ventricular end-diastolic pressure (P less than 0.05). Measurement of left ventricular contractility and relaxation, and response to beta-adrenergic stimulation were decreased in GH compared to control (P less than 0.05). Contractile protein biochemistry showed an 18% reduction in Ca2(+)-myosin ATPase activity of the left ventricle (P less than 0.05) and non-denaturing pyrophosphate gels of purified myosin demonstrated a significant shift of isoforms from the exclusive V1 pattern to both V1 and V3 isomyosins in both ventricles (P less than 0.05). In contrast to the physiological and protein biochemistry adaptations, left ventricular morphology by light microscopy and ultrastructure by electron microscopy were normal in the GH stimulated heart. There were no significant changes in myofibril fraction, in the myofibril to mitochondria ratio or in the capillary numerical density of the hypertrophied left ventricle (P = N.S.). This study demonstrates that under prolonged and extreme stimulation by GH, the heart undergoes considerable growth/hypertrophy. Although cardiac morphology remains normal during this growth, there are alterations of the isomyosins such that ATPase activity is diminished and ventricular function is decreased.
J Mol Cell Cardiol 1990 Apr
PMID:Cardiac physiology, biochemistry and morphology in response to excess growth hormone in the rat. 214 88

In order to study the mechanochemical coupling in actomyosin energy transduction, the sliding distance of an actin filament induced by one ATP hydrolysis cycle was obtained by using an in vitro movement assay that permitted quantitative and simultaneous measurements of (1) the movements of single fluorescently labeled actin filaments on myosin bound to coverslip surfaces and (2) the ATPase rates. The sliding distance was determined as (the working stroke time in one ATPase cycle, tws) x (the filament velocity, v). tws was obtained from the ATPase turnover rate of myosin during the sliding (kt), the ATP hydrolysis time (delta t) and the ON-rate at which myosin heads enter into the working stroke state when they encounter actin (kON); tws approximately 1/kt-delta t-1/kON. kt was estimated from the ATPase rates of the myosin-coated surface during the sliding of actin filaments. delta t has been determined as less than 1/100 per second, kON was estimated by analyzing the movements of very short (40 nm) filaments. The resulting sliding distance during one ATP hydrolysis cycle near zero load was greater than 100 nm, which is about ten times longer than that expected for a single attachment-detachment cycle between an actin and a myosin head. This leads to the conclusion that the coupling between the ATPase and attachment-detachment cycles is not determined rigidly in a one-to-one fashion.
J Mol Biol 1990 Nov 05
PMID:Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay. 214 98

Scallop adductor myosin is regulated by its subunits; the regulatory light chain (R-LC) and essential light chain (E-LC). Myosin light chains suppress muscle activity in the absence of calcium and are responsible for relaxation. The binding of Ca2+ to the myosin triggers contraction by releasing the inhibition imposed on myosin by the light chains. To map the functional domains of the R-LC, we have carried out mutagenesis followed by bacterial expression. Both wild-type and mutant proteins were hybridized to scallop myosin heavy chain/E-LC to map the regions of the light chain that are responsible for the binding to the myosin heavy chain/E-LC, for restoring the specific calcium-binding site, and controlling the myosin ATPase activity. The R-LC is expressed in Escherichia coli using the pKK223-3 (Pharmacia) expression vector and has been purified to greater than 90% purity. E. coli-expressed wild-type R-LC differs from the native R-LC by having the initiating methionine residue and an unblocked NH2 terminus. The wild-type R-LC restores Ca2+ binding and Ca2+ sensitivity when hybridized to scallop myosin. A point mutation of the sixth Ca2(+)-liganding position of domain I (Asp39----Ala39) results in a R-LC that binds more weakly to the heavy chain/E-LC and restores the specific Ca2(+)-binding site but not regulation of the actin-activated Mg2+ ATPase. A second mutation was produced by substituting the last 11 residues of the COOH terminus with 15 different residues. This mutant restores the specific Ca2(+)-binding site, but does not restore Ca2+ regulation to the actin-activated ATPase activity. Several other point mutations do not alter light chain function. The experiments directly establish that the divalent cation-binding site of domain I is functionally distinct from the specific Ca2(+)-binding site. The results indicate that an intact domain I and the COOH terminus are required to suppress the myosin ATPase activity. The fact that the domain I mutation and the COOH-terminal mutation disrupt regulation but do not affect Ca2(+)-binding indicates that these two aspects of regulation are separable and, therefore, the R-LC has distinct functional regions.
J Mol Biol 1990 Nov 05
PMID:Regulation of scallop myosin by mutant regulatory light chains. 214 99


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