Charles L. Stevens

Structure of the Tobacco Mosaic Virus Particle; Polymerization of Tobacco

Publisher Summary The chapter describes a critical analysis of the molecular weight of tobacco mosaic virus (TMV) and with polymerization of TMV protein. TMV has a very high molecular weight and measurements of double refraction of flow led to the conclusion that the particles are rod-shaped. The molecular weight of TMV has been determined adequately by physicochemical methods and measurements with the electron microscope. These yielded with fair uniformity a value close to 40 million. A value of about 23 A for the pitch of the helix of the virus was determined from X-ray diffraction measurements, and pertains to the virus in wet gels. In the determination of molecular weight with the electron microscope, it is desirable to utilize a technique that does not depend upon the retention of the wet particle dimensions. Some of the earliest determinations of molecular weight were done by sedimentation and diffusion. Molecular weights obtained by this method do not depend on assumptions about the molecular configuration. Light scattering has been used extensively for the determination of particle weight. The method is particularly useful because of its sensitivity over a wide range of particle sizes and its ability to provide information on particle shape. Subsequent studies have established the correctness of the assumption that water is released upon polymerization. TMV, a protein seems to be a trimer of the chemical subunit. Its polymerization has been studied primarily by light scattering and osmotic pressure methods. Within certain limits, the polymerization can be interpreted quite accurately in terms of the mathematics of condensation polymerization. Accompanying the release of water molecules upon polymerization is an increase in partial specific volume. Hydrogen ions are bound during polymerization.

Ultracentrifugation studies on early stage polymerization of tobacco mosaic virus protein

Abstract The effects of absolute temperature (T), ionic strength (μ), and pH on the polymerization of tobacco mosaic virus protein from the 4 S form (A) to the 20 S form (D) were investigated by the method of sedimentation velocity. The loading concentration in grams per liter (C) was determined at which a just-detectable concentration (β) of 20 S material appeared. It was demonstrated experimentally that under the conditions employed herein, an equilibrium concentration of 20 S material was achieved in 3 h at the temperature of the experiment and that 20 S material dissociated again in 4 h or less to 4 S material either upon lowering the temperature or upon dilution. Thus, the use of thermodynamic equations for equilibrium processes was shown to be valid. The equation used to interpret the results, log ( C−β) = constant + ( ΔH ∗ 2.3RT ) + ( Δ W ∗ el 2.3RT ) − K′sμ + ζpH, was derived from three separate models of the process, the only difference being in the anatomy of the constant; thus, the method of analysis is essentially independent of the model. ΔH ∗ and ΔW∗el are the enthalpy and the change in electrical work per mole of A protein (the trimer of the polypeptide chain), K′s is the salting-out constant on the ionic strength basis, ζ is the number of moles of hydrogen ion bound per mole of A protein in the polymerization, and R is the gas constant. The three models leading to this equation are: a simple 11th-order equilibrium between A1 (the trimer of the polypeptide chain) and D, either the double disk or the double spiral of approximately the same molecular weight, designated model A; a second model, designated B, in which A1 was assumed to be in equilibrium with D at the same time that it is in equilibrium with A2, A3, etc., dimers and trimers, etc., of A1 in an isodesmic system; and a phase-separation model, designated model C, in which A protein is treated as a soluble material in equilibrium with D, considered as an insoluble phase. From electrical work theory, Δ W el ∗ /T was shown to be essentially independent of T; therefore, in experiments at constant μ and constant pH the equation of log (C − β) versus 1/T is linear with a slope of ΔH ∗ /2.3R . The results fit such an equation over nearly a 20 °C-temperature range with a single value of Δ H ∗ of +32 kcal/mol A1. Results obtained when T and pH were held constant but μ was varied did not fit a straight line, which shows that more than simple salting-out is involved. When the effect of ionic strength on the electrical work contribution was considered in addition to salting-out, the data were interpreted to indicate a value of Δ W ∗ el of 1.22 kcal/mol A1 at pH 6.7 and a value of 4.93 for Ks′. When μ and T were held constant but pH was varied, and when allowance was made for the effect of pH changes on the electrical work contribution, a value of 1.1 was found for ζ. This means that something like 1.1 mol of hydrogen ion must be bound per mole of A1 protein in the formation of D. When this is added to the small amount of hydrogen ion bound per A1 before polymerization, at the pH values used, it turned out that for D to be formed, 1.5 H+ ions must be bound per A1 or 0.5 per protein polypeptide chain. This amounts to 1 H+ ion per polypeptide chain for half of the protein units, presumably those in one but not the other layer of the double disk or turn of the double spiral. When polymerization goes beyond the D stage, as shown by previously published data, additional H+ ions are bound. Simultaneous osmotic pressure studies and sedimentation studies were carried out, in both cases as a function of loading concentration C. These results were in complete disagreement with models A and C but agreed reasonably well with model B. The sedimentation studies permitted evaluation of the constant, β, to be 0.33 g/liter.

Equilibrium ultracentrifugation of polymers: a new method of data reduction with an application to TMV protein.

Equilibrium centrifugation is a useful method for determining stoichiometry and stability constants of associating macromolecules, and a number of such systems are of considerable biological interest. Very often, however, the most important information on the association must come from the data for which solute concentration is very low and thus relative error is high. Also, to reduce data from such experiments, absolute concentration across the cell must be known. But with the Rayleigh interference system, only concentration differences are directly obtainable. For the association of identical molecules (polymerization) where certain conditions are met, there is a function of concentration differences which appears to overcome these limitations in a straightforward way. The function can be expanded in a power series in a dimensionless positional parameter, X. The expansion has a mathematical form characteristic of the polymerization and takes particularly simple form for monomer-dimer, monomer-trimer, monomer-n-mer and indefinite (condensation) polymerization. Absolute concentration is then obtained as a parameter of the fit. If the polymerization is reversible, stability constants are directly obtainable. Using a simple graphical procedure, weightand number-average dkgree of polymerization can also be obtained, and for the latter, there is no general requirement of negligible solute concentration at the meniscus [ 1,2] . For simple polymerizations, parameters can be evaluated without complicated multivariate computer fits to sums of exponentials. The most conspicuous limitation