Calculation of the partial specific volume of proteins in concentrated salt and amino acid solutions.
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Amino Acids↗
Biomedical subjects
S N Timasheff
Publications and source records attributed to S N Timasheff.
Velocity sedimentation study of ligand-induced protein self-association.
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Computers↗
Measurement and analysis of ligand-binding isotherms linked to protein self-associations.
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Chromatography, Gel↗
Role of the dianionic form of the GTP gamma-phosphate in the polymerization process of tubulin.
To determine whether tubulin polymerization requires the bivalent metal-GTP complex with the gamma-phosphate in the dianionic form, the effect of GTP(gamma F) on the polymerization process was studied, in the presence of either magnesium or manganese. P3-fluoro P1-5'-guanosine triphosphate (GTP(gamma F)) was a competitive inhibitor (Ki = 1.8 X 10(-4) M) of the GTPase activity of tubulin-colchicine complex, stopped the polymerization process during the course of reaction and no depolymerization occurred. This indicates that GTP(gamma F) has access only to the nucleotide exchangeable site of the free tubulin dimer. Tubulin has one mole of magnesium tightly bound per mole of dimer. In order to know whether the inhibitory effect of GTP(gamma F) was due to the release of the metal, magnesium was replaced for manganese (a paramagnetic ion) and the paramagnetic effect of manganese on the fluorine NMR signal from the GTP(gamma F)-tubulin-metal complex was followed. Longitudinal and transversal relaxation rates measurements of the 1 degree F-NMR signal allowed to determine that the upper distance from the manganese site to the fluorine atom was between 6 and 8 A. These studies demonstrate that the dianionic form of the terminal phosphate of the metal-GTP complex, at the nucleotide exchangeable site, is essential to stimulate tubulin polymerization.
Guanosine 5'-O-(3-Thiotriphosphate)↗
Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding.
The preferential interactions of proteins with solvent components were studied in concentrated aqueous solutions of the sulfate, acetate, and chloride salts of Mg2+, Ba2+, Ca2+, Mn2+ and Ni2+ [except for CaSO4, BaSO4, Mn-(OAc)2, and Ni(OAc)2], and results were compared with those of the Na+ salts. It was found that, for all the salts, the preferential hydration increased in the order of Cl- less than CH3-COO- less than SO42- regardless of the cationic species used, in agreement with the anionic lyotropic series, and that the same parameter exhibited a tendency to increase in the order of Mn2+, Ni2+ less than Ca2+, Ba2+ less than Mg2+ less than Na+. The salting-out and stabilizing or salting-in and destabilizing effectiveness of the salts were interpreted in terms of the observed preferential interactions. The surface tension increment of salts, which is a major factor responsible for the preferential interactions of the Na+ salts, had no correlation with those of the divalent cation salts. It was shown that the binding of divalent cations to the proteins overcomes the salt exclusion due to the surface tension increase, leading to a decrease in the preferential hydration. In conformity with this mechanism, the preferential interaction of MgCl2 was strongly pH dependent, because of the protein charge-dependent affinity of Mg2+ for proteins, while NaCl showed no pH dependence of the preferential interaction. The proposed mechanism was supported by a strong correlation between the preferential interaction results and the interaction of these salts with the model peptide compound acetyltetraglycine ethyl ester, described by Robinson and Jencks.
Acetates↗
Protein stabilization and destabilization by guanidinium salts.
Preferential interactions of bovine serum albumin were measured with guanidine sulfate, guanidine acetate, and guanidine hydrochloride. The results showed an increasing preferential hydration with increasing salt concentration for the sulfate, positive preferential salt binding for the hydrochloride, and an intermediate situation for the acetate. These results correlate well with the known effects of the three salts on protein stability, namely, the stabilizing effect of guanidine sulfate and the denaturing effect of guanidine hydrochloride. Comparison of guanidinium and magnesium salts indicated that the substitution of guanidinium ion for Mg2+ decreases the preferential hydration and increases the preferential salt binding, suggesting that the perturbation by guanidinium ion binding of the surface free energy is greater than that by Mg2+ ion. It was concluded that guanidine salts are not a special class, but their activity toward proteins is modulated by the same fine balance between hydration and salt binding to protein as in the case of other salts, with the second factor being stronger in guanidine salts.
Acetates↗
The mechanism of action of Na glutamate, lysine HCl, and piperazine-N,N'-bis(2-ethanesulfonic acid) in the stabilization of tubulin and microtubule formation.
Preferential interaction measurements between proteins and monosodium glutamate were carried out to arrive at an understanding of the mechanism of its strong effect on tubulin stability and self-assembly into microtubules. For all proteins studied, i.e. bovine serum albumin, lysozyme, beta-lactoglobulin, and calf brain tubulin, the protein showed a large preferential hydration in the presence of monosodium glutamate. The enhancement of tubulin self-association by monosodium glutamate can be interpreted in terms of the large unfavorable free energy of interaction between the additive and the protein. Preferential interactions were also examined for lysine hydrochloride, which also gave a preferential hydration of the proteins, except for tubulin. The dependence of the preferential hydration parameter on proteins was different for the two additives, suggesting the importance of net electrostatic charges of proteins in their interaction with glutamate anions and lysinium cations. The zero preferential interaction of lysine hydrochloride with tubulin indicates an affinity of the lysine cation for the protein. Both additives increased the transition temperature of proteins. This can be understood in terms of the unfavorable free energy of interaction between the additive and the protein surface, which should be even more unfavorable when the denaturation causes an increase in the surface area.
Alkanesulfonates↗
Interaction of tubulin with bifunctional colchicine analogues: an equilibrium study.
The interaction of tubulin with simple analogues of colchicine that contain both its tropolone and trimethoxyphenyl rings has been characterized, and the results were analyzed in terms of the simple bifunctional ligand model developed for the binding of colchicine [ Andreu , J. M., & Timasheff , S. N. (1982) Biochemistry 21, 534-543] on the basis of interactions of tubulin with single-ring analogues. The compound 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6- cycloheptatrien -1-one has been found to bind reversibly to 0.86 +/- 0.06 site of purified calf brain tubulin with an equilibrium constant of (4.9 +/- 0.3) X 10(5) M-1 (25 degrees C), delta H degrees app = -1.6 +/- 0.7 kcal mol-1, and delta S degrees app = 20.5 +/- 2.5 eu. The binding appears specific for the colchicine site. The closely related compound 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino] -2,4,6- cycloheptatrien -1-one interacts weakly with tubulin. Binding of the first analogue is accompanied by ligand fluorescence appearance, quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Substoichiometric concentrations of the analogue inhibit microtubule assembly in vitro. Excess analogue concentration under microtubule-promoting conditions induces an abnormal cooperative polymerization of tubulin, similar to that of the tubulin-colchicine complex.
Animals↗
The interaction of proteins with hydroxyapatite. III. Mechanism.
The mechanism of protein binding to and elution from hydroxyapatite (HA) has been established. Binding occurs both by nonspecific attraction between protein positive charges and HA and by specific complexing of protein carboxyls with calcium loci on the mineral. Elution can take place either as the result of the nonspecific ion screening of charges or by the specific displacement of protein groups from sites on the column with which they had complexed.
Adsorption↗
Preferential interactions of proteins with solvent components in aqueous amino acid solutions.
The preferential interactions of proteins with solvent components in concentrated amino acid solutions were measured by high-precision densimetry. Bovine serum albumin and lysozyme were preferentially hydrated in all of the amino acids examined, glycine, alpha- and beta-alanine, and betaine, i.e., addition of these amino acids resulted in an unfavorable free energy change. It was shown that, for the former three amino acids, known to have a positive surface tension increment, their perturbation of the surface free energy of water is consistent with their preferential exclusion from the protein surface. In the case of betaine, which does not increase the surface tension of water, preferential exclusion from protein surface must reflect the chemical structure of this cosolvent, which is considerably more hydrophobic than that of the other three amino acids.
Amino Acids↗
Polymerization of the tubulin-colchicine complex: relation to microtubule assembly.
The polymerization of purified tubulin-colchicine complex, which results in polymers different from microtubules under microtubule-promoting conditions, has been characterized. It proceeds as a nucleated condensation polymerization, requires Mg2+, and is inhibited by small concentrations of Ca2+. Polymerization requires GTP binding, but GDP is inhibitory. The GTPase activity proceeds, but it is unlinked to polymerization. The thermodynamic characteristics of the growth reaction, namely, the apparent changes of free energy, enthalpy, entropy, heat capacity, and preferential interaction with H+ and Mg2+, are very similar to those of microtubule assembly. It is proposed that the interactions responsible for the two types of polymerization are very similar and that the molecular mechanism of microtubule inhibition by colchicine may consist in a drug-induced distortion of the normal protomer bonding geometry.
Animals↗
The interaction of vincristine with calf brain tubulin.
The interaction of the antimitotic drug vincristine with tubulin has been investigated by the techniques of self-assembly, velocity sedimentation, fluorescence, circular dichroism, and differential spectroscopy. Vincristine has been shown to inhibit the self-assembly of tubulin into microtubules at substoichiometric concentrations. The sedimentation velocity patterns at low vincristine concentration (less than 1 X 10(-5) M to 7 X 10(-5) M) consist of a bimodal boundary with a 5.8 S peak and a fast moving peak, with a nominal S20,w value of 9 S. The data conform to the ligand-promoted self-association theory of Cann and Goad (Cann, J.R., and Goad, W.B. (1972) Arch. Biochem. Biophys. 153, 603-609). At higher vincristine concentrations (greater than 8 X 10(-5 M), most of the protein is polymerized and sediments as a hypersharp peak with a nominal S20,w value of approximately 20 S. The association constant for the binding of vincristine to tubulin, determined by spectrofluorometry, is 3.5 X 10(4) liters/mol at 25 degrees C. The binding of vincristine does not induce any significant conformational changes in tubulin; however, the difference spectral results indicate perturbation of both vincristine and protein chromophores.
Animals↗
The resolution of bimodal peaks in the analytical ultracentrifuge in the presence and absence of ligands.
The patterns for peak resolution in the analytical ultracentrifuge characteristic of systems associating by different mechanisms are analyzed. These are (i) that in which reequilibration is infinitely slow; (ii) that in which reequilibration is rapid, i.e., a Gilbert-type system; and (iii) that in which reequilibration is rapid, but the self-association is mediated by a ligand. Criteria for distinguishing the three are described and illustrated by results on tubulin self-association under various conditions.
Buffers↗
Conformational states of tubulin liganded to colchicine, tropolone methyl ether, and podophyllotoxin.
The conformational effects of colchicine, podophyllotoxin, and tropolone methyl ether binding to tubulin have been studied. Conditions for the stability of the purified calf brain protein were established, and the effects of binding were examined by means of difference absorption spectroscopy, circular dichroism, fluorescence, activation of tubulin GTPase, and tubulin self-association reactions. The tubulin-colchicine complex was isolated and characterized. It displays quenched intrinsic protein fluorescence, ligand fluorescence, and GTPase activity, probably accompanied by minor perturbations in the secondary structure. The conformation of the tubulin-colchicine complex appears to be nonidentical with that of the unliganded protein. Podophyllotoxin was not found to induce any of the mentioned changes. This ligand seems to bind through a hydrophobic interaction of its trimethoxybenzene ring with tubulin, as does colchicine. Binding of tropolone methyl ether, which is the analogue of the other part of the colchicine molecule that binds to tubulin, produced effects consistent with a ligand-linked conformational change. The small perturbation by tropolone methyl ether of the circular dichroism spectrum of tubulin resembles changes induced by colchicine.
Animals↗
Stabilization of protein structure by sugars.
The preferential interaction of proteins with solvent components was measured in aqueous lactose and glucose systems by using a high precision densimeter. In all cases, the protein was preferentially hydrated; i.e., addition of these sugars to an aqueous solution of the protein resulted in an unfavorable free-energy change. This effect was shown to increase with an increase in protein surface area, explaining the protein stabilizing action of these sugars and their enhancing effect of protein associations. Correlation of the preferential interaction parameter with the effect of the sugars on the surface tension of water, i.e., their positive surface tension increment, has led to the conclusion that the surface free energy perturbation by sugars plays a predominant role in their preferential interaction with proteins. Other contributing factors are the exclusion volume of the sugars and the chemical nature of the protein surface.
Carbohydrates↗
Preferential interactions of proteins with salts in concentrated solutions.
The preferential interactions of proteins with solvent components were studied in concentrated salt by densimetric measurements. Proteins were found to be preferentially hydrated in NaCl, NaCH3COO, and Na2SO4. The resulting unfavorable free-energy change was related to the effects of these salts on solubility and stability of the proteins. This unfavorable free-energy change was correlated with the large, positive surface tension increment of these salts, i.e., their perturbation of surface free energy. On the other hand, KSCN, CaCl2, and MgCl2 showed considerable binding to bovine serum albumin, which could be related to their destabilizing and salting-in effects on macromolecules. Since the last two salts have high surface tension increments, it was concluded that this does not necessarily lead to protein preferential hydration and stabilization.
Mathematics↗
Aging of tubulin at neutral pH.
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Animals↗
In vitro vinblastine-induced tubulin paracrystals.
Purified calf brain tubulin has been induced to self-aggregate in vitro into paracrystalline structures by the anti-cancer drug vinblastine. The size, shape, optical birefringence, and drug stoichiometry of these tubulin-vinblastine paracrystals are similar to those of paracrystals formed in vivo. Similar structures can be formed with vincristine and desacetylvinblastine, but not with colchicine or podophyllotoxin.
Animals↗