Ragaa A. Shalaby

Hydrogen ion uptake upon tobacco mosaic virus protein polymerization.

Abstract To determine the stage at which H+ ions are bound during the entropy-driven polymerization of tobacco mosaic virus protein, acid-base titrations were carried out at a concentration of 5 mg/ml in 0.1 m -KCl from pH 8 to pH 5.2 and back to pH 8 at 4, 10, 15 and 20 °C. The titration was always completely reversible when the addition of acid or base was so slow that the experiment required seven hours in each direction. When the titration was started at pH 7 and performed down and up twice as rapidly, a hysteresis loop, indistinguishable from one previously published, was obtained at 20 °C. Ultracentrifugation experiments were carried out at selected pH values at the four temperatures. H+ ion uptake, as determined from the reversible titration curves, is correlated with the disappearance of the 4 S component and is independent of whether the polymerized species is in a 20 S or higher state of aggregation. At pH 7, approximately 1 mole of H+ ion is bound per mole of monomer. At pH values between 6.56 and 6.05, 1.5 moles of H+ ion are bound per mole of monomer upon polymerization. At pH 6.05, 0.5 mole of H+ ion is bound before any polymerization takes place. Tobacco mosaic virus protein at 20 °C in an unbuffered 0.1 m -KCl solution at pH 7.18 at a concentration of 41 mg/ml, largely in the 20 S state, was depolymerized entirely to the 4 S state by dilution with 0.1 m -KCl adjusted to the same pH. Under these conditions, there was no pH change, indicating that no H + ions are released. These seemingly contradictory findings can be explained by assuming that the 4 S component polymerizes to form either double discs without binding H+ ions, or, alternatively, two-turn helices accompanied by the binding of H+ ions. Both double discs and two-turn helices sediment at approximately 20 S. Whether polymerization in the neighborhood of pH 7 leads to helices or discs depends upon the availability of H+ ions.

The effect of ionic strength on the entropy-driven polymerization of tobacco mosaic virus protein: Contributions of electrical work and salting-out☆

Abstract The entropy-driven polymerization of tobacco mosaic virus protein is favored by an increase in ionic strength, μ, and by a decrease in pH. The effect of ionic strength is interpreted in terms of salting-out and electrical work, a function of charge and, therefore, of pH as well as of μ. The extent of polymerization is measured in terms of a characteristic temperature, T ∗ , corresponding to a characteristic value of the equilibrium constant, K c T∗ is measured at an early stage in the polymerization process where the optical density increment from light scatter is 0.01. The theory developed encompassing both salting-out and electrical work terms relates 1 T ∗ to μ approximately according to the equation, 1 T ∗ = C + Bμ − A μ 1 2 , where C is the ratio of entropy to enthalpy, B is proportional to the salting-out constant divided by enthalpy, and A μ 1 2 depends upon the square of the charge and is proportional to the electrical work contribution divided by the enthalpy. Data in which μ varied from 0.025 to 0.150 at three pH values, 5.95, 6.35, and 6.50, were fitted to this equation and the parameters C , B , and A were evaluated. Experiments were also carried out at a constant μ of 0.10 at pH values in increments of 0.1 between 5.9 and 6.8. The theory predicts that, at constant μ, 1 T ∗ , corrected for the electrical work contribution, is a linear function of pH with a negative slope proportional to the number of hydrogen ions bound per protein unit during polymerization, divided by the enthalpy. The data obtained fit two straight lines with different slopes above and below pH 6.3. Independent experiments carried out by the method of Stevens and Loga show that the number of hydrogen ions bound per protein unit also differs above and below pH 6.3 and the ratio of these is the same as the ratio of the above mentioned slopes. The data, therefore, make it possible to evaluate the enthalpy to be 24.8 kcal/mol of associating A protein and, with this value, the parameters C , B , and A can be interpreted. Standard entropies range from 86 e.u. at pH 6.5 to 88.5 at pH 5.95 and the salting-out constant, K S ′ , is 2.2 at all pH values studied. At μ = 0.10, the values of the electrical work contribution at pH 5.95, 6.35, and 6.50 are +0.298, +0.455, and +0.534 kcal/mol, respectively. Theoretical calculations from models predict values in agreement within a factor of less than two.

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.

Entropy-driven polymerization of protein from the E66 strain of tobacco mosaic virus☆

Abstract Protein of the tobacco mosaic virus mutant E66 has lysine replacing asparagine of the type strain, vulgare, at position 140. Thus, E66 protein should have one more positive or one less net negative charge than vulgare at pH 6 to 7. To investigate the effect of charge, a comparative study of the polymerization of E66 and vulgare proteins at pH 6.0, 6.2, 6.4, 6.6, and 6.8 at ionic strengths 0.15, 0.10, and 0.05 was made by turbidimetry. Polymerization of E66 protein always proceeded at a lower temperature than vulgare. However, the extent of polymerization was much lower in E66, especially at the higher ionic strengths. Sedimentation velocity results paralleled those from turbidity measurements in that E66 protein polymerizes at lower temperatures than vulgare; the 20 S component is more abundant in E66 protein. Osmotic pressure measurements also show that E66 protein is more polymerized than vulgare, especially at lower pH values. Hydrogen ion titrations of E66 protein were carried out from pH 8 to 5 and back to pH 8 in 0.10 m KCl at three temperatures, 4, 10, and 15 °C. These titrations were reversible when carried out slowly. The isoionic point is near pH 5; thus the charge at pH 7.5 is −3. The reversible titration results were correlated with the aggregates present at the various pH values and temperatures, determined from the areas under the schlieren peaks in sedimentation velocity experiments. It is found that hydrogen ion binding at the three pH values is correlated with the disappearance of the smallest aggregates and is independent of the type of higher polymer formed. To investigate the effect of ionic strength and pH on the characteristic temperature corresponding to an optical density increment of 0.01 by the method used previously for vulgare, two sets of turbidity measurements were carried out. In the first one the ionic strength was changed from 0.025 to 0.15 in increments of 0.025 at pH 6.0 and 6.4. In the other set, the ionic strength was kept constant at 0.10 and the pH changed from 5.9 to 6.7 in increments of 0.1 pH units. When the analysis of these data was carried out, Δ H ∗ = 30 kcal/mol was obtained. For the salting out constant a value of 1.7 was found, compared to 2.2 for vulgare, a result consistent with the fact that E66 should be less hydrophobic than vulgare. The electrical work term ΔWel also turns out to be about one-half that for vulgare, which is expected from the lower net negative charge on E66 protein.

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

Abstract 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.

Entropy-driven polymerization of the protein from the flavum strain of tobacco mosaic virus.

Abstract Protein of the flavum mutant of tobacco mosaic virus has the same number of positive groups as vulgare but aspartic acid at position 19 in vulgare is replaced by alanine, yielding the same net charge as E66 protein, that is, one less negative charge near pH 7 than vulgare protein. Comparative temperature-OD measurements of both flavum and vulgare proteins, prepared identically, were made at pH 5.8 to 6.6 at ionic strengths of 0.10 and 0.05. Another set of T-OD experiments was carried out at two pH values, 5.9 and 6.2, with the ionic strength varying from 0.025 to 0.15 in increments of 0.025. Compared to vulgare, flavum protein polymerizes at higher temperatures and exhibits qualitatively similar effects of pH and ionic strength. Hydrogen ion titrations were carried out from pH 8 to pH 5 and back to pH 8 in 0.10 m KCl at three temperatures, 4, 10, and 15 °C. The titration was completely reversible when carried out slowly. The isoionic point is about pH 5; the charge at pH 7.5 is −3. The hydrogen ion binding was correlated with the different aggregates present under different conditions of pH and temperatures as determined from sedimentation velocity measurements. It was found that H + ion binding correlates with the disappearance of the smallest aggregates present and does not depend on the type of higher polymer formed. From the analysis for the effect of ionic strength and pH on the characteristic temperature, T ∗ , defined as the temperature corresponding to an optical density increment of 0.01, values of Δ H ∗ , Δ S ∗ , K s ′, and Δ W el are obtained. The salting out constant, K s′ , for flavum protein turns out to be considerably higher than for vulgare and E66 proteins. This is to be expected of a protein with a higher content of hydrophobic residues. In contrast, the electrical work term turns out to be close to the value obtained for E66 protein and about one-half of the value for vulgare, consistent with the lower net negative charge.

Comparison of the entropy-driven polymerization reactions of E66 and vulgare tobacco mosaic virus proteins.

The effects of temperature (T), ionic strength (mu), and pH on the polymerization of the coat protein of the E66 strain of tobacco mosaic virus (TMV) from the 4 S form (A), a trimer of the polypeptide chain, to the 20 S form (D) were investigated by the method of sedimentation velocity. Interpretations of thermodynamic parameters were based on only those data obtained in experiments for which reversibility could be demonstrated both by lowering temperature and by lowering concentration. E66 protein differed from vulgare TMV in that, in position 140, lysine replaced asparagine. Thus, E66 protein should be less hydrophobic than vulgare protein and Ks, the salting-out constant, should be less. The charge on unpolymerized E66 protein was -3 proton units per polypeptide chain, compared to -4 for vulgare protein. The electrical work contribution, delta W*el, for E66 protein should be (-3/-4)2, or 0.5625 that for vulgare. The results were that delta W*el at pH 6.7, 15 degrees C, and mu = 0.1 was 0.700 kcal/mol for E66 protein compared to 1.22 for vulgare. The experimental ratio was 0.574; Ks = 2.16 for E66 compared to 4.93 for vulgare. Hydrogen ions (1.5) were bound per A unit, or 0.5 per polypeptide chain, in the formation of D from A. delta H*, the enthalpy change per mole of A, was 33 kcal at pH 6.7 and 36 at pH 6.9, compared to 30 at both pH values for vulgare protein. delta S*, the entropy change per mole of A, was +132.1 e.u. for E66 compared to 127.4 for vulgare. Entropy-driven processes are found in dynamic biological situations. Ready reversibility at biological temperatures is a requirement, yet the polymer structures must be strong and well ordered. This is achieved through a large number of weak bonds between subunits, combined with ready reversibility under slightly changed conditions. The significant role of water is to facilitate depolymerization by binding to subunits.

Effect of dipolar ions on the entropy-driven polymerization of tobacco mosaic virus protein☆

The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 degrees C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein.

Polymerization of tobacco mosaic virus protein without and with hydrogen ion binding

When tobacco mosaic virus (TMV) protein is polymerized at pH values above 7 in unbuffered solutions, either by raising temperature at constant ionic strength or by increasing ionic strength at constant temperature, a 20 S component is formed having bound only the very small amount of H+ ion supplied by the unpolymerized protein. When hydrogen ion is added by titration during polymerization so as to keep pH constant, as would occur automatically if a buffer were present, a 20 S component is formed with one H+ ion bound each for half of the subunits. Thus, a 20 S form with and a 20 S form without bound H+ ion exist. Furthermore, the 20 S form without bound H+ ion binds H+ ion when supplied by titration to produce a 20 S form with the same amount of bound H+ ion as when H+ ion is supplied during the polymerization.

Entropy-driven polymerization of ribgrass virus protein

Holmes ribgrass virus (HRV), because of serological results, is regarded as a distantly related strain of tobacco mosaic virus (TMV). HRV protein differs substantially in amino acid sequence from TMV protein, especially in that it contains one histidine residue and three methionine residues, compared to none of either for TMV protein. Ultracentrifugation and hydrogen ion titration data on HRV protein, similar to those obtained previously for the early stage polymerization of TMV and E66 proteins, demonstrated some similarities and more distinct differences from those of the other two proteins. The major similarities are that the early polymerization of HRV protein is entropy driven and the first major polymerized product is a 20 S component, presumably a double disk or two-turn helix, as in the case of the other proteins. The major differences are that the unpolymerized HRV protein sediments at 3 S rather than at the 4 S for the others; it is presumably a dimer of the polypeptide chain. The enthalpy of polymerization per mole of A protein, delta H*, is 18,400 cal for HRV protein, compared to about 30,000 for TMV protein. One mol of H+ ion/mol HRV A protein, compared to 1.5 for TMV and E66 proteins, is bound during polymerization to the 20 S state. Contrasted with the other proteins, very little if any electrical work contribution was detected for the HRV protein. A major difference was found in hydrogen ion titration. Unpolymerized HRV protein binds hydrogen ions significantly in the unpolymerized A protein state, unlike the A proteins from the other two viruses.