Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule.

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.     

A model of stereocilia adaptation based on single molecule mechanical studies of myosin I

We have used an optical tweezers-based apparatus to perform single molecule mechanical experiments using the unconventional myosins, Myo1b and Myo1c. The single-headed nature and slow ATPase kinetics of these myosins make them ideal for detailed studies of the molecular mechanism of force generation by acto-myosin. Myo1c exhibits several features that have not been seen using fast skeletal muscle myosin II. (i) The working stroke occurs in two, distinct phases, producing an initial 3 nm and then a further 1.5 nm of movement. (ii) Two types of binding interaction were observed: short-lived ATP-independent binding events that produced no movement and longer-lived, ATP-dependent events that produced a full working stroke. The stiffness of both types of interaction was similar. (iii) In a new type of experiment, using feedback to apply controlled displacements to a single acto-myosin cross-bridge, we found abrupt changes in force during attachment of the acto-Myo1b cross-bridge, a result that is consistent with the classical 'T2' behaviour of single muscle fibres. Given that these myosins might exhibit the classical T2 behaviour, we propose a new model to explain the slow phase of sensory adaptation of the hair cells of the inner ear.     

Reaction of myosin with salicylaldehyde I. The effect of salicylaldehyde on the physicochemical properties of myosin

The specificity and nature of the reaction between salicylaldehyde and myosin and the effect of salicylalation on the molecular parameters of myosin were studied. The following observations were made.

1. 1. The reaction of salicylaldehyde with the lysyl residues of myosin is specific, since no salicylaldehyde is bound if the lysyl residues of myosin are trinitrophenylated.

2. 2. Salicylaldehyde is bound by myosin through the formation of an azomethine linkage (Schiff's base). This was established from the measured difference absorption spectrum of the myosin-salicylaldehyde complex.

3. 3. Three groups of lysyl residues can be distinguished with respect to the reaction with salicylaldehyde, namely, (a) residues with high association constant (K_ass = 1.8 ± 0.9·10⁵ M^-1), (b) residues with moderate association constant (K_ass = 2.2·10³ M^-1) and (c) residues that react with salicylaldehyde only after the denaturation of the protein. Their numbers could be estimated as 10 ± 5, 130 ± 5 and 260 ± 5 per mole myosin, respectively. The first group of residues was found to be absent from heavy and light meromyosin, the proteolytic fragments of myosin.

4. 4. The reaction is reversible. The complex formation rate constant, evaluated from the formula for second order reaction, is 2.2 sec^-1·M^-1, and the decomposition rate constant for first order reaction is 1.1·10^-3 sec^-1 at 22°.

5. 5. The reaction is pH dependent, the reaction yield increasing at higher pH.

6. 6. The solubility of myosin at low ionic strength decreases with increasing degree of salicylalation at slightly alkaline pH.

7. 7. The intrinsic viscosity of myosin does not change on salicylalation.

8. 8. A second peak due to polymerization appears on the sedimentation profile of the protein if more than 70 lysyl residues are salicylalated per mole of myosin.

Subunit interactions of lobster hemocyanin. I. Ultracentrifuge studies

Subunit interactions in the hemocyanin of New England lobster, Homarus americanus, were investigated by means of the ultracentrifuge, using sedimentation velocity and Archibald molecular weight methods. It was verified that a 17S species dimerizes rapidly and reversibly to form a 25S species in the pH range 9.4–9.7 in the presence of calcium ion. From the Ca²⁺ and pH dependence of the equilibrium constant for this process, the absorption of approximately five calcium ions and three protons accompany the formation of one molecule of the 25S species. The sedimentation velocity patterns were also found to shift in favor of the 17S species with the imposition of excess hydrostatic pressure.     

Rabbit cardiac myosin. I. Physical and chemical characterization of the native molecule.

Rabbit cardiac myosin, isolated from frozen tissue, was effectively purified by batchwise treatment with DEAE-cellulose in addition to suing cilution-precipitation techniques. An extensive experimental program was subsequently carried out with respect to the enzymic amino acid, optical and physicochemical properties of native cardiac myosin. This program has included the following: examination of the effects of pH and varying concentrations of ATP, CaCl2, MgCl2, and PCMB on its ATPase activity; measurement of its circular dichroic spectrum in solvent buffers, at different pH or containing ATP in the absence or presence of Ca-2+ or Mg-2+ ions; study of the concentration dependence of its viscosity and sedimentation velocity at low temperatures; and investigation of its molecular weight by the Archibald method and low- and high-speed sedimentation equilibrium. The results of these studies were consistent with the interpretation that cardiac myosin is comprised of highly asymmetric, semi-rigid molecules with a molecular weight in the order of 4.7 times 10-5, which display non-ideality even in solvent buffers of high ionic strength at neurtal pH. In addition, computer analysis of the high-speed sedimentation equilibrium data has provided evidence for the presence of a self-association reaction at low protein concentration. Even though the specif ATPase activity of cardiac myosin was found to be approximately one-third that reported for skeletal myosin in all cases, it was concluded, on the the basis of the essentially analogous physical and chemical properties of rabbit cardiac and skeletal myosin, that the two proteins are very similar in terms of molecular size, shape, and secondary structure.     

Comparative studies on myosin ATPase of a flying and nonflying bird.

Myosin was purified from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird. Ki (ADP) of myosin ATPase of pigeon is higher, but the Km (ATP) is lower than that of fowl. The specific activity (mumole of Pi liberated/min/mg protein) is higher for the fowl. A0.5 (CaCl2) of myosin of both pigeon and fowl is similar. However, the two proteins differ in their interactions with ADP, ATP and p-chloromercuribenzoate. The two proteins have the same tyrosine, tryptophan and sulfhydryl contents. The electrophoretic patterns of the two myosins on SDS-polyacrylamide gels are different. These studies show significant molecular differences in the myosin derived from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird.     

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.     

Substructure of the myosin molecule. 3. Preparation of single-headed derivatives of myosin

Native myosin has two globular regions attached to an a-helical rod. Papain is able to cleave the globular “heads” from the rod, leading to the formation of a variety of single-headed molecules. Among these subfragments are isolated globules (HMM S-1) and single globules attached to helical rods of lengths varying from 500 to 1400 Å. These subfragments can be separated from the other products of the proteolytic digestion by salt elution from a DEAE-cellulose column. Some of the properties of single-headed heavy meromyosin and myosin have been determined by hydrodynamic methods, and shadow-cast preparations of these subfragments have been directly visualized by electron microscopy. In addition to providing further evidence for the presence of two similar halves in myosin, these new subfragments can be used in studies related to the question of why myosin has two active “heads”.     

Segmental flexibility in the myosin molecule: Evidence from binding studies of myosin fragments with actin

From comparative studies of the association with polymeric actin of the bifunctional species heavy meromyosin and its monofunctional constituents, information about the relative freedom of these paired elements can be derived. An isotherm for the former binding process is presented which involves, as an experimentally determinable parameter, the local concentration of a second segment after the first of a pair is attached to the lattice. From combined data for these two association reactions a value of 10⁻⁴ M is obtained for this quantity. The large degree of segmental flexibility reported for the free heavy meromyosin is still manifested in the association with actin.