Protein precipitation and denaturation by dimethyl sulfoxide.

Solvent conditions play a major role in a wide range of physical properties of proteins in solution. Organic solvents, including dimethyl sulfoxide (DMSO), have been used to precipitate, crystallize and denature proteins. We have studied here the interactions of DMSO with proteins by differential refractometry and amino acid solubility measurements.

Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects.

Arginine has been used to suppress protein aggregation and protein-protein or protein-surface interactions during protein refolding and purification. While its biotechnology applications are gradually expanding, the mechanism of these effects of arginine has not been fully elucidated. Arginine is more effective at higher concentrations, an indication of weak interactions with the proteins.

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.

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.

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.

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.

Modulation of recombinant human prostate-specific antigen: activation by Hofmeister salts and inhibition by azapeptides. Appendix: thermodynamic interpretation of the activation by concentrated salts.

Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators.

In disperse solution, "osmotic stress" is a restricted case of preferential interactions.

In the practice of "osmotic stress," the effect of excluded cosolvents on a biochemical equilibrium is interpreted as the number of water molecules participating in the reaction. This action is attributed to lowering of solvent water activity by the cosolvent. This concept of osmotic stress in disperse solution is erroneous: (i) A cosolvent cannot be both excluded and inert, i.

Role of the colchicine ring A and its methoxy groups in the binding to tubulin and microtubule inhibition.

The roles of the methoxy substituents on ring A of two ring colchicine (COL) analogues were probed by the synthesis of a number of drugs and the examination of their effect on binding to tubulin, inhibition of microtubule assembly, and induction of GTPase activity. Selective elimination of ring A methoxy groups at positions 2, 3, and 4 weakened all three processes. The effects on binding and inhibition were independent of the nature of ring C (or C').

Linkages in tubulin-colchicine functions: the role of the ring C (C') oxygens and ring B in the controls.

Linkages between structural components of colchicine (COL) and its biphenyl analogues (allocolchicine, ALLO, and its analogues) in the binding to tubulin and its functional consequences were scrutinized. Three ring ALLO analogues with the carbomethoxyl in position 4' of ring C' replaced by a carbomethyl (KAC) and methoxy (MAC) groups were synthesized. The binding properties and consequences of binding (microtubule inhibition, abnormal polymerization, and induction of GTPase activity) were compared within the series of three ring and two ring compounds, as well as between pairs consisting of a two ring and a three ring compound with identical groups in position 4'.

The thermodynamic mechanism of protein stabilization by trehalose.

The stabilization of ribonuclease A by alpha-alpha-trehalose was studied by preferential interaction and thermal unfolding. The protein is stabilized by trehalose at pH 2.8 and pH 5.

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.

Structural analysis of the substoichiometric and stoichiometric microtuble-inhibiting biphenyl analogues of colchicine.

The structures of the colchicine (COL) analogues, 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB)and 2,3,4,4'-tetramethoxy-1,1'-biphenyl (TMB), were solved by X-ray diffraction. Their comparison with the structure of colchicine indicated the ability of both compounds to enter into a colchicine binding pocket. Comparison of TKB with 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) showed that the methyl group of the carbomethoxy group in position 4' of TCB protrudes beyond the (C=O)-CH3 group in the same position in TKB.

Stoichiometric and substoichiometric inhibition of tubulin self-assembly by colchicine analogues.

The mechanism of the stoichiometric and substoichiometric inhibitions of tubulin self-assembly by several structural analogues of colchicine (COL) was investigated. The inhibition data were analyzed in terms of a simple model that takes into consideration Kg, the normal microtubule growth constant, equal to Cr-1 (Cr is the critical concentration for microtubule formation), and Kb, the binding constant of the drug to tubulin. In this manner, the value of the microtubule inhibition constant (Ki), which is the binding constant of the tubulin-drug complex to the end of a growing microtubule (which stops the microtubule growth), was determined.

On the role of surface tension in the stabilization of globular proteins.

The stabilization of proteins by a variety of co-solvents can be related to their property of increasing the surface tension of water. It is demonstrated that, during the thermal unfolding of proteins, this increase of the surface tension can be overcome by the increase in the temperature of the solution at the midpoint of the transition, Tm, and the weak binding of co-solvent molecules. Three such co-solvents were studied: trehalose, lysine hydrochloride (LysHCl), and arginine hydrochloride (ArgHCl).

Contribution of the surface free energy perturbation to protein-solvent interactions.

Surface tension measurements were carried out at 20 degrees C by a capillary drop-weight method on aqueous solutions of sodium glutamate (NaGlu), lysine hydrochloride (LysHCl), potassium aspartate (KAsp), arginine hydrochloride (ArgHCl), lysylglutamate (LysGlu), argininylglutamate (ArgGlu), guanidinium sulfate, trehalose, trimethylamine N-oxide (TMAO), dimethyl sulfoxide, 2-methyl-2,4-pentanediol (hexylene glycol), and poly(ethylene glycol)s of molecular weights 200, 400, 600, and 1000. All of the salts and the sugar increased the surface tension of water, while the last four compounds decreased it, with 2-methyl-2,4-pentanediol lowering it most effectively and TMAO being the least effective. The preferential hydration of bovine serum albumin (BSA) and lysozyme was measured in KAsp, ArgHCl, LysGlu, and ArgGlu.

Why do some organisms use a urea-methylamine mixture as osmolyte? Thermodynamic compensation of urea and trimethylamine N-oxide interactions with protein.

Many organisms accumulate low molecular weight substances known as osmolytes when they experience environmental water stress. The main classes of osmolytes are sugars, polyhydric alcohols, amino acids and their derivatives, and methylamines, and all are known to be protein stabilizers. However, marine cartilaginous fishes and the coelacanth use, as osmolytes, a combination of urea and methylamines, i.

Cooperative multiple binding of bisANS and daunomycin to tubulin.

The binding of daunomycin and bisANS to tubulin was studied by direct equilibrium techniques. Both ligands generated abnormal Scatchard plots. Their concave-downward nature indicated positive cooperativity.

Energy transfer studies of the distances between the colchicine, ruthenium red, and bisANS binding sites on calf brain tubulin.

Fluorescence energy transfer experiments were performed in order to measure the spatial separation between the colchine and Ruthenium Red binding sites, the high-affinity bisANS and Ruthenium Red sites, and the allocolchicine and high-affinity bisANS sites on calf brain tubulin. Energy transfer was observed between both colchicine and allocolchicine and Ruthenium Red, resulting in a distance of 40-45 A between these sites on the tubulin molecule. No detectable energy transfer could be observed when allocolchicine was used as fluorescence donor and bisANS as acceptor or when bisANS was used as donor and Ruthenium Red as acceptor.

Cosolvent modulation of the tubulin-colchicine GTPase-activating conformational change: strength of the enzymatic activity.

The locus of action of cosolvent additives in the activation of the tubulin-colchicine GTPase was investigated. The GDP off rates were slowed down by the cosolvents in a manner that parallels their specific viscosities, indicating that diffusion-controlled release of GDP may be rate-limiting under the conditions of these studies. Yet, the net effect of cosolvents was to increase the overall rate of GTP hydrolysis.

The colchicine-induced GTPase activity of tubulin: state of the product. Activation by microtubule-promoting cosolvents.

Colchicine induces a weak assembly-independent GTPase activity in calf brain tubulin [David-Pfeuty, T., Erickson, H. P.