Structures and roles of the polymorphic forms of tobacco mosaic virus. V. Conservation of the abnormally titrating groups in tobacco mosaic virus

The two groups per subunit which titrate with an abnormal pK of about 7 in tobacco mosaic virus Vulgare have also been found in three other naturally occurring strains and two mutants. Possession of these groups, almost certainly carboxyl-carboxylate pairs, therefore appears to be a crucial feature of the virus protein structure, which has been conserved during the evolution of the strains. The residues responsible may be narrowed down to those carboxylic acid residues which are at the same position in all the tobacco mosaic virus variants titrated. Residues 115 and 116 make up one probable pair, while residue 145 and one other, as yet unidentified, make up the second probable pair.     

Structures and roles of the polymorphic forms of tobacco mosaic virus protein. 8. Initial stages of assembly of nucleoprotein rods from virus RNA and the protein disks

The initial stages of the assembly of tobacco mosaic virus have been investigated by reassembling the RNA with a radioactively labelled protein disk preparation and then completing the reaction by the addition of a large excess of an unlabelled disk preparation. This gives measurements of the numbers of subunits incorporated at early times and the growth curves have been plotted.These curves have been analysed in terms of a bimolecular nucleation reaction, which is first order in the disk concentration, with a rate constant of 1.3 × 10³ mol^?1 s^?1, and then an elongation which saturates at high protein concentrations to a maximum rate of 7.6 subunits s^?1, with a K_m of 0.84 mg/ml for the disk preparation.These kinetic parameters, and the predicted overall assembly curves, have been compared with data previously determined by other methods and agree closely, showing that the different experimental techniques give consistent results. The measurements are fully compatible with our earlier hypotheses Butler &; Klug 1971 that the nucleation with virus RNA and protein disks is rapid compared with the subsequent rod elongation and that this elongation can occur most rapidly directly from the protein disks. They are not compatible with the contention of some other workers that elongation cannot occur directly from disks, but only from the smaller A-protein.     

Structures and roles of the polymorphic forms of tobacco mosaic virus protein. VII. Lengths of the growing rods during assembly into nucleoprotein with the viral RNA

The length distributions of growing particles have been determined and followed as a function of time during the reconstitution of tobacco mosaic virus from its isolated RNA and protein. The protein was supplied either largely as the “disk” aggregate or as A-protein obtained by cooling a disk preparation. In a further experiment, the growth was initiated with disks and then continued with A-protein. It has been possible to correct the resulting distributions of lengths for the effect of broken RNA molecules and hence to obtain a picture of the distribution of lengths of the growing particles.From these distributions and also the average lengths, it is concluded that the growth is most rapid when disks are the protein source, giving full length particles in six to ten minutes. When A-protein is supplied for the growth, the rate is about one quarter of that with disks, irrespective of whether the rods have been nucleated with disks or not.     

Structures and roles of the polymorphic forms of tobacco mosaic virus protein: VI. Assembly of the nucleoprotein rods of tobacco mosaic virus from the protein disks and RNA

The assembly of tobacco mosaic virus involves a preformed protein aggregate, the disk, which consists of two rings each of 17 protein subunits, as the sole protein source. The kinetics of this assembly have been studied, using both tobacco mosaic virus RNA, which causes a rapid initiation and so enables growth to be studied, and also polyadenylic acid, with which initiation is slowed down and thus can be partially resolved from growth. Two disks interact with a special nucleotide sequence at the 5′-hydroxyl end of a single tobacco mosaic virus RNA molecule to initiate the formation of the viral nucleoprotein helix, which then grows by the addition of further disks. All of the subunits from these further disks are incorporated into the helix, so that growth proceeds by the co-operative addition of 34 subunits at a time. Under the conditions used, rearrangement of each disk takes about six seconds, giving a total time for the growth of a complete virus particle of just over six minutes.     

Letter: Hysteresis of proton binding to tobacco mosaic virus protein associated with metastable polymerization.

Proton binding to tobacco mosaic virus protein at 20 °C has been found to exhibit a reproducible hysteresis which results from the metastability of high molecular weight helical, virus-like rods. In a titration from pH 4 or 5 to 7, the time for depolymerization of such rods, as measured by ultracentrifugation, decreases from days to minutes over a range of about a tenth of a pH unit, near pH 6·6 at 20 °C. Relative to the extent of proton binding in the depolymerized state at 4 °C, the magnitude of the hysteresis near pH 6·2 corresponds to more than 50% of the protons bound per subunit in the equilibrium polymerized state.     

Calcium ion binding by isolated tobacco mosaic virus coat protein

Calcium ion titrations were performed on solutions of tobacco mosaic virus coat protein using a calcium-specific ion-exchange electrode. Isolated coat protein was found incapable of binding calcium ions under equilibrium conditions at pH values above its iso-ionic point (pH 4.3 to 4.6). However, calcium ions were found to bind to coat protein under non-equilibrium conditions, which suggests that the isolated coat protein has the proper conformation to bind calcium ions at the iso-ionic point.     

Kinetics of thermal aggregation of tobacco mosaic virus coat protein

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52°C in a wide range of protein concentrations, [P]₀. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52°C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]₀ (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42°C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]₀ and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates {A/A _lim; t} (t is time and A _lim is the limiting value of A at t ). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.     

Calcium ion binding by tobacco mosaic virus

Calcium ion titrations were performed on solutions of tobacco mosaic virus using a calcium-specific ion-exchange electrode. Scatchard analyses were used to obtain the number of calcium ion binding sites per protein subunit (n) and the apparent stability constant for complex formation (beta' Ca). These experiments were performed on unbuffered solutions, in either water or 0.01 M-KCl, to allow a determination of the number of hydrogen ions released per calcium ion bound (chi). The results indicate that near neutrality, the virus particle possesses two calcium ion binding sites per subunit having apparent stability constants greater than 10(4) M-1. The results are interpreted as if these two sites are non-identical and titrate independently. The higher affinity site for the virus in water has a value of log beta' Ca, which varies from about 8.5 at pH 8.5 to about 3.9 at pH 5.0, and for the virus in 0.01 M-KCl has a value that varies from about 6.2 at pH 8.0 to about 3.7 at pH 5.5. The higher affinity site for the virus in water binds up to two competing hydrogen ions, one with an apparent pKH value greater than 8.5 and the other with a value that varies from 6.0 at pH 5.5 to 7.3 at pH 8.0. For the virus in 0.01 M-KCl, only the competing hydrogen ion binding with an apparent pKH value greater than 8.5 remains. The results could be interpreted as indicating that the electrical charge on the virus particle has a constant value in the pH range 5.5 to 8.0 despite the fact that hydrogen ion titration curves for the intact virus particle indicate that the charge should vary from about -1 per subunit at pH 5.5 to about -4 at pH 8.0.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering

The kinetics of heat-induced and cetyltrimethylammonium bromide induced amorphous aggregation of tobacco mosaic virus coat protein in Na(+)/Na(+) phosphate buffer, pH 8.0, have been studied using dynamic light scattering. In the case of thermal aggregation (52 degrees C) the character of the dependence of the hydrodynamic radius (R(h)) on time indicates that at certain instant the population of aggregates is split into two components. The size of the aggregates of one kind remains practically constant in time, whereas the size of aggregates of other kind increases monotonously in time reaching the values characteristic of aggregates prone to precipitation (R(h)=900-1500 nm). The construction of the light scattering intensity versus R(h) plots shows that the large aggregates (the start aggregates) exist in the system at the instant the initial increase in the light scattering intensity is observed. For thermal aggregation the R(h) value for the start aggregates is independent of the protein concentration and equal to 21.6 nm. In the case of the surfactant-induced aggregation (at 25 degrees C) no splitting of the aggregates into two components is observed and the size of the start aggregates turns out to be much larger (107 nm) than on the thermal aggregation. The dependence of R(h) on time for both heat-induced aggregation and surfactant-induced aggregation after a lapse of time follows the power law indicating that the aggregation process proceeds in the kinetic regime of diffusion-limited cluster-cluster aggregation. Fractal dimension is close to 1.8. The molecular chaperone alpha-crystallin does not affect the kinetics of tobacco mosaic virus coat protein thermal aggregation.