Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.     

Negative staining of myosin molecules

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.     

Unshadowed myosin molecules: STEM mass-maps of myosin heads.

Myosin molecules were directly visualized without heavy metal shadowing by scanning transmission electron microscopy (STEM) under low dose conditions. The general appearance and dimensions of heavy metal-free molecules were similar to those of shadowed myosin, either after freeze-drying without or air-drying with glycerol. Two characteristic configurations of myosin head regions were found, a first type showing two pear-shaped heads with narrow necks and a second type showing two heads connected by an extra mass in the central regulatory domain where the light chains are located. The mass of the latter type (mol. wt. = 265 +/- 39 kd) is in excellent accordance with biochemical data whereas the mass of the first type is somewhat lower (mol. wt. 219 +/- 44 kd).     

Two non-identical heads of myosin molecules

Four different preparations of skeletal subfragment-1, denoted in this report as S1(Aa), S1(Ab), S1(Ba), and S1(Bb), and two different preparations of cardiac subfragment-1, denoted as S1(A) and S1(B), were obtained as described in our recent report (J. Biochem. 97, 965, 1985). (i) The four preparations were obtained from chicken breast myosin trinitrophenylated with 2,4,6-trinitrobenzene sulfonate in the absence of inorganic pyrophosphate (-PPi), and they were all shown to be trinitrophenylated. Addition of PPi caused change in the absorption spectra of trinitrophenyl(TNP)-S1(Aa) and TNP-S1(Ba), but not in those of TNP-S1(Ab) and TNP-S1(Bb). (ii) The two preparations of S1 were obtained from cardiac myosin trinitrophenylated either in the absence (-) or presence (+) of PPi. S1(B) was trinitrophenylated, whereas S1(A) was not. Specifically emphasized is the observation that the yield of cardiac S1(A) was practically equal to that of cardiac S1(B). On the basis of these results, we propose the hypothesis of "two iso-myosins with non-identical heads," which is essentially a combination of the hypothesis of isoenzymes and that of non-identical heads.     

Packing of myosin molecules in muscle thick filaments

The backbone of the myosin filament is an aggregate of alpha-helical coiled coil myosin rods. Its surface forms a three-stranded helix composed of myosin heads. Currently there is no adequate model to describe the organization of the myosin filament. It is proposed here that, in cross-section the light meromyosin (LMM) of 18 myosin molecules form an outer tube, with nine S2 forming the interior core. At the surface of the thick filament, myosin heads are arranged in three rows, giving the filament a periodicity of 14.3 nm per three myosin molecules. Two of these molecules are organized at an angle of 120 degrees to each other on the same level, while the third is shifted 7.2 nm along the filament axis. This packing gives a striation pattern of 7.2 nm by electron microscopy. An alternative model is also possible, in which the heads of the myosin molecules are uniformly spaced at an interval of 14.3 nm along the filament axis. The packing of individual molecules within the myosin filament is based on a regular pattern of charge on the 28 amino-acid repeat in the rod domain.     

Regulation of asymmetric smooth muscle myosin II molecules

The emerging view of smooth/nonmuscle myosin regulation suggests that the attainment of the completely inhibited state requires numerous weak interactions between components of the two heads and the myosin rod. To further examine the nature of the structural requirements for regulation, we engineered smooth muscle heavy meromyosin molecules that contained one complete head and truncations of the second head. These truncations eliminated the motor domain but retained two, one, or no light chains. All constructs contained 37 heptads of rod sequence. None of the truncated constructs displayed complete regulation of both ATPase and motility, reinforcing the idea that interactions between motor domains are necessary for complete regulation. Surprisingly, the rate of ADP release was slowed by regulatory light chain dephosphorylation of the truncated construct that contained all four light chains and one motor domain. These data suggest that there is a second step (ADP release) in the smooth muscle myosin-actin-activated ATPase cycle that is modulated by regulatory light chain phosphorylation. This may be part of the mechanism underlying "latch" in smooth muscle.     

Biological peculiarities of someYersinia species: strain-dependent virulence and strain-dependent stress proteins

A pair ofYersinia enterocolitica serotype O∶3 strains — incubated in growth medium with 10% NaCl and 2 mM glycin betaine at 4 °C, were used to study the plasmid role in the infection of BALB/c mice. The isogenic plasmid-bearing strain, but not its plasmidless derivative, caused enteric infection and histological changes in intestines, stomach and liver of the mice. Two strainsY. enterocolitica andYersinia pseudotuberculosis were incubated at different temperatures (4 and 25 °C) in media, supplemented with different concentrations of NaCl. Two concentrations of betaine as osmoprotector were tested. The initial strains and their substrains were characterised by protein profiles using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Results show that in salinity conditions, the presence of osmoprotectant (betaine) as well as the temperature of cultivation plays a great role in the expression of some bacterial proteins. The manner in which the different strains answer to the stress situations is specific for each of them.     

Transport of actin-decorated liposomes along myosin molecules in vitro

We examined whether actin filaments bound to positively charged liposomes could interact with myosin molecules and induce liposome motility. When liposomes were constructed from the mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cationic N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), actin filaments bound to the liposomes. The actin-bound liposomes exhibited movement on myosin molecules in the presence of adenosine-5'-triphosphate (ATP). The displacement was almost linearly increased with time and the behavior differed from that of Brownian motion. Furthermore, the presence of 30% DOTAP in liposomes was most effective for transport. These data show that the actomyosin system was successfully integrated into the liposomes and possesses the ability to actively transport useful agents enclosed within the liposomes.     

Direct observation of processive movement by individual myosin V molecules

Myosin V is an unconventional myosin thought to move processively along actin filaments. To have hard evidence for the high processivity, we sought to observe directly the movement by individual native chick brain myosin V (BMV) molecules with fluorescent calmodulin. Single BMV molecules did exhibit highly processive movement along actin filaments fixed to a coverslip. BMV continued to move up to the barbed end of its actin track, and did not readily detach from action. The barbed end, therefore, got brighter with time, because of a constant stream of BMV traffic. The maximum speed of the processive movement was 1 microm/s, and the maximum actin-activated ATPase rate was 2.4 s(-1). These values apparently imply that BMV travels a great distance, 400 nm, per an ATPase cycle.     

Visualization of freeze-dried and shadowed myosin molecules immobilized on electron microscopic films

The mica replication technique first described by Hall [5] has produced myosin molecules which were heterogeneous in appearance in terms of shadowing, decoration, contrast and background. Therefore, an alternative technique for the visualization of myosin molecules was developed: Myosin molecules are sprayed directly onto glow discharged or silicium-monoxide coated carbon filmed grids, omitting glycerol. After washing several times with distilled water, rapid freezing, and freeze-drying, the immobilized myosin molecules are visualized by shadow-casting at low temperature and at varying angles. After backing with carbon the "in situ" shadowed molecules are observed in the electron microscope. This technique has several advantages over the standard method in that it yields more reproducible results. It is potentially useful for investigating interactions of myosin binding proteins with myosin and for visualizing unshadowed myosin in the STEM.     

Cryo-atomic force microscopy of unphosphorylated and thiophosphorylated single smooth muscle myosin molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.     

Mechanical properties of single myosin molecules probed with the photonic force microscope

To characterize elastic properties and geometrical parameters of individual, whole myosin molecules during their interaction with actin we sparsely adsorbed myosin molecules to nanometer-sized microspheres. Thermally driven position fluctuations of these microspheres were recorded with the three-dimensional detection scheme of the photonic force microscope. Upon binding of single myosin molecules to immobilized actin filaments in the absence of ATP, these thermally driven position fluctuations of the microspheres change significantly. From three-dimensional position fluctuations stiffness and geometrical information of the tethering molecule can be derived. Axial stiffness was found to be asymmetric, approximately 0.04 pN/nm for extension, approximately 0.004 pN/nm for compression. Observed stiffness of whole myosin molecules is much less than estimated for individual myosin heads in muscle fibers or for single-molecule studies on myosin fragments. The stiffness reported here, however, is identical to stiffness found in other single-molecule studies with full-length myosin suggesting that the source of this low stiffness is located outside the myosin head domain. Analysis of geometrical properties of tethering myosin molecules by Brownian dynamics computer simulations suggests a linker length of approximately 130 nm that is divided by a free hinge located approximately 90 nm above the substrate. This pivot location coincides with myosin's hinge region. We demonstrate the general applicability of thermal fluctuation analysis to determine elastic properties and geometrical factors of individual molecules.     

The physical chemistry of cruciform structures in supercoiled DNA molecules

Inverted repeat DNA sequences extrude cruciform structures when present in negatively supercoiled molecules, stabilised by the release of torsional stress brought about by the negative twist change. We have revealed the presence of cruciform structures by means of enzyme and chemical probing experiments and topological band shift methods. The geometry of cruciform structures has been studied from two points of view. The unpairing of bases in the loop region has been investigated using bisulphite modification, with the result that the central four nucleotides have single-stranded character, and the next pair have only partially single-stranded nature. Gel electrophoretic studies of a pseudo-cruciform structure indicate that the cruciform junction introduces a pronounced bend into the molecule. The dependence of the formation of the ColE1 cruciform upon DNA supercoiling shows that it has a free energy of formation of 18.4 +/- 0.5 kcal mole-1. The kinetics of the extrusion process are complex. Most sequences extrude slowly with considerable temperature coefficients, but the detailed properties are strongly sequence-dependent. One synthetic inverted repeat sequence which we have studied in detail has an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole-1. We discuss possible mechanistic pathways for the extrusion process.     

DNA-templated click chemistry for creation of novel DNA binding molecules

We have developed a new methodology for producing new molecules that bind to dsDNA using DNA-templated click chemistry. The click reactions between the minor groove binding peptide and acridine intercalators were accelerated by the addition of dsDNA. Furthermore, the resulting peptide–acridine conjugate showed a slightly stronger binding to dsDNA. These results indicate that the DNA-templated click chemistry is applicable for screening new binding molecules.