Ligand effects on protein thermodynamic stability

A simple, partition-function formalism is used to describe the coupling between ligand binding and protein equilibrium unfolding. This general theoretical framework is shown to provide an adequate basis for the analysis of experimental ligand effects on the unfolding of complex protein systems. Nevertheless, the most important consequences of ligand binding for protein thermodynamic stability, as exposed by the partition-function approach, are found to be those demonstrated by Julian Sturtevant about 20 years ago.     

Allowance for effects of thermodynamic nonideality in sedimentation equilibrium distributions reflecting protein dimerization

This reexamination of a high-speed sedimentation equilibrium distribution for α-chymotrypsin under slightly acidic conditions (pH 4.1, I(M) 0.05) has provided experimental support for the adequacy of nearest-neighbor considerations in the allowance for effects of thermodynamic nonideality in the characterization of protein self-association over a moderate concentration range (up to 8 mg/mL). A widely held but previously untested notion about allowance for thermodynamic nonideality effects is thereby verified experimentally. However, it has also been shown that a greater obstacle to better characterization of protein self-association is likely to be the lack of a reliable estimate of monomer net charge, a parameter that has a far more profound effect on the magnitude of the measured equilibrium constant than any deficiency in current procedures for incorporating the effects of thermodynamic nonideality into the analysis of sedimentation equilibrium distributions reflecting reversible protein self-association.     

Direct allowance for the effects of thermodynamic nonideality in the quantitative characterization of protein self-association by osmometry

A procedure is described for the direct analysis of osmotic pressure data for reversibly dimerizing proteins that makes allowance for effects of thermodynamic nonideality on the statistical–mechanical basis of the potential-of-mean-force between molecules. Detailed consideration is also given to calculation of the magnitudes of the required virial coefficients. After illustration of the approach with analysis of simulated osmotic pressure data, the method is used to obtain dimerization constants from published osmotic pressure data for soybean proteinase inhibitor, hemoglobin and α-chymotrypsin.     

Allowance for thermodynamic nonideality in the characterization of protein interactions by spectral techniques

Theory is developed for the characterization of protein interactions by spectral techniques, where the constraints of constant temperature and pressure demand that thermodynamic activity be defined on the molal concentration scale. The customary practice of defining the equilibrium constant (K) on a molar basis is accommodated by developing expressions to convert those experimental values (K_molar) to their thermodynamically more rigorous counterparts (K_molal). Such procedures are illustrated by reanalysis of published results for the effects of molecular crowding agents on the isomerisation of α-chymotrypsin and reversible complex formation between catalase and superoxide dismutase. Although those reanalyses have led to only minor refinements of the quantitative interpretation, it is clearly preferable to adopt thermodynamic rigor throughout future spectral studies by employing the molal concentration scale from the outset.     

Salt-tolerance in plants. I. Ions, compatible organic solutes and the stability of plant ribosomes

Abstract Polysomes and ribosomes recovered from a number of plant species were tested for stability when incubated at 25°C in salt solutions in the absence of ATP and initiation factors. Stability was assessed by sucrose density gradient analysis. The stability was inversely proportional to salt concentrations above 125 mol m⁻³ KCl. Polysomes were less stable in the presence of Na⁺ than K⁺ salts, and were much less stable in Cl⁻ than in acetate salts. Polysomes from Triticum aestivum. Hordeum vulgare, Capsicum annuum, Helianthus annuus. Pisum sativum, Atriplex nummularia, Beta vulgaris, Cladophora sp., Enteromorpha sp. and Corallina cuvieri were similarly sensitive to KCl. Polysomes from Ulva lactuca were more sensitive than the other species. Cytoplasmic and plastid polysomes from T. aestivum were similarly unstable in 500 mol m⁻³ KCl. Unprogrammed ribosomal subunit couples from T. aestivum, B. vulgaris and U. lactuca showed Mg²⁺-dependent conformational instability and dissociation in KCl. Slight differences in ribosomal stability were observed between species, but these were unrelated to the salt tolerances of the plants. The ‘compatible’ organic solutes, glycinebetaine and proline, failed to reduce ion-induced instability. Ribosome yield and polysome profiles were similar in leaves of B. vulgaris containing significantly different levels of both Na⁺ and Cl⁻ after growth in media containing 50 or 200 mol m⁻³ NaCl. The results are consistent with the hypothesis that plants maintain a cytoplasmic solute environment that is compatible with ribosomal stability.     

Effects of thermodynamic nonideality in kinetic studies

Experimental evidence is presented for concentration dependence of the pseudo-firstorder rate constant describing the rate of inversion of sucrose by 2 m HCl; and also of the increase in maximal velocity for the catalytic reduction of pyruvate by lactate dehydrogenase that results from addition of the inert macromolecular solutes bovine serum albumin, ovalbumin, and Dextran T70. These somewhat unusual and seemingly diverse observations are examined in terms of a theory formulated on the basis of two equilibrium reactions, the first describing complex formation between two reactants, and the second isomerization of that complex to an activated state prior to product formation. This formulation permits consideration of activity coefficient ratios relevant to the equilibria and the expression of these ratios as power series in total solution composition. Quantitative assessment of the experimental results is made possible in these terms by estimating the magnitudes of the constant coefficients of the virial expansions as excluded volumes. It is concluded that the result observed in the sucrose inversion study finds rational explanation in thermodynamic nonideality factors governing the overall equilibrium between the reactants and the activated complex of sucrose and hydronium ion. For the enzyme-catalyzed reaction the same general equation applies but particular attention is given to the simplified form that is relevant to high substrate concentrations, where, in the absence of inert compounds, the conventional maximal velocity is approached. In this region an increase in velocity observed upon addition of an inert macromolecular component may be considered explicitly in terms of excluded volume effects related to a shape change in the isomerization between enzyme-substrate complex and its activated state.     

Analysis of thermodynamic non-ideality in terms of protein solvation.

The effects of thermodynamic non-ideality on the forms of sedimentation equilibrium distributions for several isoelectric proteins have been analysed on the statistical-mechanical basis of excluded volume to obtain an estimate of the extent of protein solvation. Values of the effective solvation parameter delta are reported for ellipsoidal as well as spherical models of the proteins, taken to be rigid, impenetrable macromolecular structures. The dependence of the effective solvated radius upon protein molecular mass exhibits reasonable agreement with the relationship calculated for a model in which the unsolvated protein molecule is surrounded by a 0.52-nm solvation shell. Although the observation that this shell thickness corresponds to a double layer of water molecules may be of questionable relevance to mechanistic interpretation of protein hydration, it augurs well for the assignment of magnitudes to the second virial coefficients of putative complexes in the quantitative characterization of protein-protein interactions under conditions where effects of thermodynamic non-ideality cannot justifiably be neglected.     

Distribution of compatible solutes in the halophilic methanogenic archaebacteria.

Accumulation of compatible solutes, by uptake or de novo synthesis, enables bacteria to reduce the difference between osmotic potentials of the cell cytoplasm and the extracellular environment. To examine this process in the halophilic and halotolerant methanogenic archaebacteria, 14 strains were tested for the accumulation of compatible solutes in response to growth in various extracellular concentrations of NaCl. In external NaCl concentrations of 0.7 to 3.4 M, the halophilic methanogens accumulated K+ ion and low-molecular-weight organic compounds. beta-Glutamate was detected in two halotolerant strains that grew below 1.5 M NaCl. Two unusual beta-amino acids, N epsilon-acetyl-beta-lysine and beta-glutamine (3-aminoglutaramic acid), as well as L-alpha-glutamate were compatible solutes among all of these strains. De novo synthesis of glycine betaine was also detected in several strains of moderately and extremely halophilic methanogens. The zwitterionic compounds (beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine) and potassium were the predominant compatible solutes among the moderately and extremely halophilic methanogens. This is the first report of beta-glutamine as a compatible solute and de novo biosynthesis of glycine betaine in the methanogenic archaebacteria.     

Effects of thermodynamic nonideality in ligand binding studies

Effects of thermodynamic nonideality are considered in relation to the quantitative characterization of the interaction between a small ligand. S, and a macromolecular acceptor. A, by two types of experimental procedure. The first involves determination of the concentration of ligand in dialysis equilibrium with the acceptor/ligand mixture, and the second, measurement of the concentration of unbound ligand in the reaction mixture by ultrafiltration or the rate of dialysis method. For each situation explicit expressions are formulated for the appropriate binding function with allowance for composition-dependent nonideality effects expressed in terms of molar volume, charge-charge interaction and covolume contributions. The magnitudes of these effects are explored with the aid of experimental studies on the binding of tryptophan and of methyl orange to bovine serum albumin. It is concluded for experiments conducted utilizing either equilibrium dialysis or frontal gel chromatography that, provided a correction is made for any Donnan redistribution of ligand, theoretically predicted acceptor-concentration dependence is likely to be negligible and that use of the conventional binding equation written for an ideal system is appropriate to the analysis of the results. Use of ultrafiltration or the rate of dialysis method requires examination of the assumption that the activity coefficient ratio y(A)y(s)/y(AS) for the reaction mixture approximates unity; but again reassurance is provided that nonideality manifested as a dependence of the binding function on acceptor concentration is unlikely to be significant.     

Effects of thermodynamic nonideality on protein interactions. Equivalence of interpretations based on excluded volume and preferential solvation

Published results on the stabilization of proteins by sucrose (J.C. Lee and S.N. Timasheff, J. Biol. Chem. 256 (1981) 7193) have been reexamined and interpreted in terms of thermodynamic nonideality. The composition dependence of activity coefficients may be accounted for on a statistical-mechanical basis using the concept of excluded volume. An expression is derived in which the effect of sucrose on determination of the partial specific volume of a protein, previously interpreted in terms of preferential protein solvation, is also seen to be attributable to excluded volume. Gel chromatographic studies of the reversible unfolding of alpha-chymotrypsin are presented which demonstrate temperature- and sucrose-mediated changes in the effective volume of the enzyme. These measurements support the quantitative interpretation of the stabilization in terms of thermodynamic nonideality arising from the difference between covolumes for sucrose and the two isomeric states of alpha-chymotrypsin. By establishing the equivalence of the two approaches that have been used to account for the effects of inert solutes on protein transitions, the present investigation eliminates the need for any distinction between such solutes on the basis of molecular size; and also enhances greatly the potential sensitivity of thermodynamic nonideality as a means of probing protein isomerizations, since greater displacement of the equilibrium position may be effected by small rather than by macromolecular solutes present at the same weight concentrations.     

The effect of compatible solutes on proteins

Summary The ability of the compatible solute, proline, to affect the behavior of proteins has been examined in many different systems by many researches. In the present study of protein solvation, proline has been shown to prevent or diminish, in a concentration-dependent manner, the glutamine synthetase-precipitating ability of polyethylene glycol (PEG). The effects of PEG concentration and molecular weight are reduced by proline, and the interaction is strongly affected by pH.PEG causes precipitation of many proteins, and the ability of proline to reduce the precipitation of two non-enzymatic conjugated proteins, alfalfa mosaic virus and an³H-testosterone/antiserum complex, was also examined. Proline was effective in reducing the PEG-induced precipitation of both proteins. Virus precipitation by PEG and its alleviation by proline are influenced by pH. The increased virus-precipitating effect of PEG in the presence of salt (NaCl) is also alleviated by proline. The precipitation of the radioimmune complex by PEG is diminished by proline and by a mixture of free amino acids.These results indicate the generality of the three-way interaction between proline, protein and PEG. They may be of importance for extraction of proteins from biological systems and in studies of enzyme inactivation or protein denaturation in a cytoplasmic milieu. The results suggest that the protective effects of some amino acids are at least additive and are consistent with the conclusion that the compatible solutes protect protein-containing systems against the unfavorable consequences of dehydration and other stresses, by increasing the tendency of the system to maintain thestatus quo.     

Properties of compatible solutes in aqueous solution

We have performed Molecular Dynamics simulations of ectoine, hydroxyectoine and urea in explicit solvent. Special attention has been spent on the local surrounding structure of water molecules. Our results indicate that ectoine and hydroxyectoine are able to accumulate more water molecules than urea by a pronounced ordering due to hydrogen bonds. We have validated that the charging of the molecules is of main importance resulting in a well defined hydration sphere. The influence of a varying salt concentration is also investigated. Finally we present experimental results of a DPPC monolayer phase transition that validate our numerical findings.     

中度嗜盐菌相容性溶质机制的研究进展

生活在高盐环境中的中度嗜盐菌不仅能抗衡外界的高渗透压胁迫,而且还能迅速适应短时间内的渗透冲击。为适应该环境,中度嗜盐菌依赖于一种被称为相容性溶质的物质,以执行渗透保护功能。这类物质属于极性的、易溶的和低分子量的有机化合物,其中包括糖类、氨基酸类、甜菜碱类和四氢嘧啶类等。中度嗜盐菌主要采用相容性溶质机制来适应盐环境。在此,就中度嗜盐菌的盐适应机理、相容性溶质的种类和特点,以及其作用的分子机制进行了阐述和讨论。     

Unusual deprivation of compatible solutes in Acinetobacter baumannii

The opportunistic human pathogen Acinetobacter baumannii is one of the leading causes of nosocomial infections. The high prevalence of multidrug-resistant strains, a high adaptability to changing environments and an overall pronounced stress resistance contribute to persistence and spread of the bacteria in hospitals and thereby promote repeated outbreaks. Altogether, the success of A. baumannii is mainly built on adaptation and stress resistance mechanisms, rather than relying on ‘true’ virulence factors. One of the stress factors that pathogens must cope with is osmolarity, which can differ between the external environment and different body parts of the human host. A. baumannii ATCC 19606^T accumulates the compatible solutes glutamate, mannitol and trehalose in response to high salinities. In this work, it was found that most of the solutes vanish immediately after reaching stationary phase, a very unusual phenomenon. While glutamate can be metabolized, mannitol produced by MtlD is excreted to the medium in high amounts. First results indicate that A. baumannii ATCC 19606^T undergoes a rapid switch to a dormant state (viable but non-culturable) after disappearance of the compatible solutes. Resuscitation from this state could easily be achieved in PBS or fresh medium.     

Diversity and biosynthesis of compatible solutes in hyper/thermophiles.

The accumulation of compatible solutes, either by uptake from the medium or by de novo synthesis, is a general response of microorganisms to osmotic stress. The diversity of compatible solutes is large but falls into a few major chemical categories, such as carbohydrates or their derivatives and amino acids or their derivatives. This review deals with compatible solutes found in thermophilic or hyperthermophilic bacteria and archaea that have not been commonly identified in microorganisms growing at low and moderate temperatures. The response to NaCl stress of Thermus thermophilus is an example of how a thermophilic bacterium responds to osmotic stress by compatible solute accumulation. Emphasis is made on the pathways leading to the synthesis of mannosylglycerate and glucosylglycerate that have been recently elucidated in several hyper/thermophilic microorganisms. The role of compatible solutes in the thermoprotection of these fascinating microorganisms is also discussed.     

New compatible solutes related to Di-myo-inositol-phosphate in members of the order Thermotogales.

The accumulation of intracellular organic solutes was examined in six species of the order Thermotogales by nuclear magnetic resonance spectroscopy. The newly discovered compounds di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate and di-myo-inositol-1,3'-phosphate were identified in Thermotoga maritima and Thermotoga neapolitana. In the latter species, at the optimum temperature and salinity the organic solute pool was composed of di-myo-inositol-1,1'(3,3')-phosphate, beta-glutamate, and alpha-glutamate in addition to di-myo-inositol-1,3'-phosphate and di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate. The concentrations of the last two solutes increased dramatically at supraoptimal growth temperatures, whereas beta-glutamate increased mainly in response to a salinity stress. Nevertheless, di-myo-inositol-1,1'(3,3')-phosphate was the major compatible solute at salinities above the optimum for growth. The amino acids alpha-glutamate and proline were identified under optimum growth conditions in Thermosipho africanus, and beta-mannosylglycerate, trehalose, and glycine betaine were detected in Petrotoga miotherma. Organic solutes were not detected, under optimum growth conditions, in Thermotoga thermarum and Fervidobacterium islandicum, which have a low salt requirement or none.     

Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes

Microorganisms respond to osmotic stress mostly by accumulating compatible solutes, either by uptake from the medium or by de novo synthesis. These osmotically active molecules preserve the positive turgor pressure required for cell division. The diversity of compatible solutes is large but falls into a few major chemical categories; they are usually small organic molecules such as amino acids or their derivatives, and carbohydrates or their derivatives. Some are widely distributed in nature while others seem to be exclusively present in specific groups of organisms. This review discusses the diversity and distribution of known classes of compatible solutes found in prokaryotes as well as the increasing knowledge of the genes and pathways involved in their synthesis. The alternative roles of some archetypal compatible solutes not subject to osmoregulatory constraints are also discussed.     

Accumulation of cyclitols functioning as compatible solutes in the haptophyte alga Pavlova sp.

Using nuclear magnetic resonance spectroscopy, we identified and characterized accumulated compatible solutes in cells of the haptophyte alga Pavlova sp. strain CCMP504. The predominant organic solutes were d ‐1,4/2,5‐cyclohexanetetrol (CHT), 1,3,5/2,4‐cyclohexanepentol (CHP) and scyllo‐inositol. We then profiled the intracellular organic solutes present in Pavlova sp. grown in medium with salinity ranging from 23 practical salinity units (PSU) (hyposaline) to 35 PSU (optimum salinity for growth), to 47 PSU (hypersaline). The results of these analyses reveal progressive accumulation of CHT and CHP in response to increasing growth medium salinity. We also observed altered accumulation of CHT and CHP in samples subjected to salinity shock. To further characterize the CHT and CHP biosynthesis in Pavlova sp., we carried out stable isotope feeding experiments. Specific labeling of CHT and CHP with d ‐¹³C‐glucose suggested that d ‐glucose is a biosynthetic precursor of these cyclitols. The salinity‐induced accumulation of CHT and CHP suggests that these cyclitols act as compatible solutes. Our results therefore provide new evidence supporting classification of CHP as a compatible solute.     

Elimination of self-association as the source of the thermodynamic nonideality in aqueous proline solutions

The effect of high concentrations of proline on the diffusion coefficient of water has been examined to assess the extent to which the resulting thermodynamic nonideality could be explained on the statistical-mechanical basis of excluded volume. In fact, such a space-filling role not only accounts for the proline concentration-dependence of the diffusion coefficient of water but it also accounts for the nonideality of proline in freezing point depression and isopiestic measurements. These findings refute the conclusion (Schobert, B. and Tschesche, H. (1978) Biochim. Biophys. Acta 541, 270–277) that the stabilization of enzyme structure by high concentrations of proline stems from self-association of the imino acid via intermolecular hydrogen bonding; and thereby support the concept that the protective effect of proline on enzyme stability must reside mainly in its action as an inert, space-filling solute.