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Serge N Timasheff

Publications and source records attributed to Serge N Timasheff.

5 recordsLinked to original sources
PubMed2003

Preferential interactions of urea with lysozyme and their linkage to protein denaturation.

The interactions involved in the denaturation of lysozyme in the presence of urea were examined by thermal transition studies and measurements of preferential interactions of urea with the protein at pH 7.0, where it remains native up to 9.3 M urea, and at pH 2.0, where it undergoes a transition between 2.5 and 5.0 M urea. The destabilization of lysozyme by urea was found to follow the linear dependence on urea molar concentration, M(u), DeltaG(u)(o)=DeltaG(w)(o)-2.1 M(u), over the combined data, where DeltaG(u)(o) and DeltaG(w)(o) are the standard free energy changes of the N right harpoon over left harpoon D reaction in urea and water, respectively. Combination with the measured preferential binding gave the result that the increment of preferential binding, deltaGamma(23)=Gamma(23)(D)-Gamma(23)(N), is also linear in M(u). A temperature dependence study of preferential interactions permitted the evaluation of the transfer enthalpy, DeltaHmacr;(2,tr)(o), and entropy, DeltaSmacr;(2,tr)(o) of lysozyme from water into urea in both the native and denatured states. These values were found to be consistent with the enthalpy and entropy of formation of inter urea hydrogen bonds (Schellman, 1955; Kauzmann, 1959), with estimated values of DeltaHmacr;(2,tr)(o)=ca. -2.5 kcal mol(-1) and DeltaSmacr;(2,tr)(o)=ca. -7.0 e.u. per site. Analysis of the results led to the conclusion that the stabilization of the denatured form was predominantly by preferential binding to newly exposed peptide groups. Combination with the knowledge that stabilizing osmolytes act by preferential exclusion from peptide groups (Liu and Bolen, 1995) has led to the general conclusion that both the stabilization and destabilization of proteins by co-solvents are controlled predominantly by preferential interactions with peptide groups newly exposed on denaturation.

Binding Sites↗
PubMed2003

Polymerization and calcium binding of the tubulin-colchicine complex in the GDP state.

The tubulin-colchicine complex instead of tubulin was used in an imidazole buffer throughout experiments. The interaction with calcium was examined, especially in the GDP state. The high affinity sites of calcium took part in the polymerization of the complex in the GTP state, while the low ones participated in the depolymerization. The complex had 2 high affinity sites with the dissociation constant of 11.5 x 10(-6) M, and 16 low affinity sites with the dissociation constant of 2.27 x 10(-4) M in the GTP state. In the case of GDP state, the dissociation constant of the high affinity site was 7.2 x 10(-6) M, and the low affinity site was not observed. The ultracentrifugal experiment indicated a little compact structure in the GTP state compared with the GDP state. This agreed with the results of calcium binding.

Animals↗
PubMed2002

Thermodynamic binding and site occupancy in the light of the Schellman exchange concept.

An analysis of Schellman's treatment of preferential interactions is presented, as viewed by a laboratory practitioner of the art. Starting with an intuitive description of what binding is in terms of the distribution of molecules of water and of a weakly interacting ligand (co-solvent), Schellman proceeded to a rigorous thermodynamic definition in which he showed that classical, dialysis equilibrium, binding is a purely thermodynamic quantity. Putting water and the co-solvent on an equivalent footing, he showed that the classical binding treatment is inadequate for weakly interacting systems, in which the replacement of water by ligand and exclusion of co-solvent are symmetrical concepts. Analyzing specifically the simple model of a single independent site, Schellman demonstrated how a positive binding constant can give rise to a measured negative binding stoichiometry. He showed that the origin of the complicated binding isotherms is the non-idealities of water and co-solvent, and went further to analyze critically the effect of site heterogeneity on the ligand concentration dependencies of site occupancy, preferential binding and the thermodynamic quantities, enthalpy, entropy and Gibbs free energy. This exposition of the Schellman treatment is accompanied by illustrations drawn from the experimental results obtained in this author's laboratory.

Binding Sites↗
PubMed2002

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components.

Solvent additives (cosolvents, osmolytes) modulate biochemical reactions if, during the course of the reaction, there is a change in preferential interactions of solvent components with the reacting system. Preferential interactions can be expressed in terms of preferential binding of the cosolvent or its preferential exclusion (preferential hydration). The driving force is the perturbation by the protein of the chemical potential of the cosolvent. It is shown that the measured change of the amount of water in contact with protein during the course of the reaction modulated by an osmolyte is a change in preferential hydration that is strictly a measure of the cosolvent chemical potential perturbation by the protein in the ternary water-protein-cosolvent system. It is not equal to the change in water of hydration, because water of hydration is a reflection strictly of protein-water forces in a binary system. There is no direct relation between water of preferential hydration and water of hydration.

Binding Sites↗