Self-association In Solution Research Articles

Studies of solute self-association by sedimentation equilibrium: allowance for effects of thermodynamic non-ideality beyond the consequences of nearest-neighbor interactions

A sedimentation equilibrium study of α-chymotrypsin self-association in acetate-chloride buffer, pH 4.1 I 0.05, has been used to illustrate determination of a dimerization constant under conditions where thermodynamic non-ideality is manifested beyond the consequences of nearest-neighbor interactions. Because the expressions for the experimentally determinable interaction parameters comprise a mixture of equilibrium constant and excluded volume terms, the assignment of reasonable magnitudes to the relevant virial coefficients describing non-associative cluster formation is essential for the evaluation of a reliable estimate of the dimerization constant. Determination of these excluded volume parameters by numerical integration over the potential-of-mean-force is shown to be preferable to their calculation by approximate analytical solutions of the integral for this relatively small enzyme monomer with high net charge (+10) under conditions of low ionic strength (0.05 M).

A functional assay in Escherichia coli to detect non-assisted interaction between galactose repressor dimers.

Among the Escherichia coli operons repressed from multiple sites on DNA, the galactose operon is unique: its repression requires an auxiliary protein, HU, to assist cooperative repressor binding to two distant DNA sites. Here we show that GalR can still mediate repression from distant sites in an artificial and simplified regulatory region which totally disturbs the organisation of the natural interactions. This simple and unexpected cooperation of a protein incapable of self-association in solution might be involved in regulation of the gal operon. Furthermore, the assay may be generalised to detection of rather weak cooperative interactions between DNA-bound proteins.

A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I

A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I

A Pro→Ala substitution in melittin affects self-association, membrane binding and pore-formation kinetics due to changes in structural and electrostatic properties

Melittin, the main component of bee venom of Apis mellifera, contains a proline at position 14, which is highly conserved in related peptides of various bee venoms. To investigate the structural and functional role of Pro14 a melittin analogue was studied where proline is substituted by an alanine residue (P14A). The investigations were focussed on: (i) the secondary structure in aqueous solution and membranes; (ii) the self-association in solution; (iii) the binding to POPC membranes; and (iv) the P14A-induced leakage and pore formation in membrane vesicles. Circular dichroism and gel filtration experiments showed that P14A exists at concentrations <12 μM in monomeric form with an α-helicity of 28±7%. A further increase in peptide concentration leads to the formation of large aggregates consisting of 9±1 monomers. While binding studies with POPC vesicles revealed for P14A a stronger binding affinity towards membranes than for melittin, the peptide-induced leakage of fluorescent markers from vesicles was less efficient for P14A than for melittin. Furthermore, an unexpected efflux behaviour at high values of bound P14A was observed which indicated that the pore formation kinetics for P14A is more complex than it was reported for melittin. The different features of P14A in aggregation, binding and efflux compared to melittin are mainly ascribable directly to structural changes caused by the proline→alanine substitution. Furthermore, the results indicate an improved screening of the positively charged residues of P14A by counterions which contributes additionally to the observed differences in peptide activities. It is suggested that the presence of proline in melittin is not only of structural importance but also influences indirectly the electrostatic properties of the native peptide.

Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops.

Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.

Hydrophobic and solvation effects on the solubility of hydroxysteroids in various solvents Quantitative and qualitative assessment by application of the mobile order and disorder theory

Following the preliminary discussion outlining the relative difficulty of the experimental determination of solubilities versus the lack of general applicability and sound theoretical basis of most current predictive approaches for solubility, we look closely at the recently developed pure thermodynamic model for solubility in real solutions: that derived from mobile order and disorder theory. With successful estimates of the solubility of 62hydroxysteroids and related drugs in common organic solvents of differing polarities and H-bonding capacity, the model has proved to be a valuable tool in predicting the solubility of complex solutes such as polyfunctional drugs. Free of any adjusted regression coefficients and based on a limited number of readily available parameters, the proposed model is a time-saving alternative procedure to experimentation. By properly quantifying the enthalpic and entropic contributions involved in the overall solubility process, the model furthermore assesses the factors that determine solubility differences between steroids and solubility changes upon solvent properties. Therefore, the poor solubility of hydroxysteroids in aliphatic hydrocarbons results from the negative effects due to the change in non-specific cohesion forces upon mixing and due to steroid self-association in solution. In water, the low solubilities are mainly due to the large negative value of the hydrophobic effect which cannot be overcome by steroid–water functional group associations, i.e., the solvation effect. The relatively good solubility of hydroxysteroids in polar non-associated solvents (ketones, ethers, esters) and in alcohols is explained by the fact that, in both kinds of solvents, steroid self-association is rather well counterbalanced by the formation of a more or less important number of steroid–solvent interactions without being penalized by a strong negative hydrophobic effect in the case of alcohols. Some practical rules regarding how some parameters like the molar volume or the substitution may affect solubility are finally derived, which might help the pharmaceutical scientist to orient the choice of a solvent for liquid pharmaceutical forms.

From gel filtration to biosensor technology: the development of chromatography for the characterization of protein interactions.

The objective of this review is to summarize the development of chromatographic techniques for the determination of reaction stoichiometries and equilibrium constants for solute interactions of biological importance. Gel chromatography is shown to offer a convenient means of characterizing solute self-association as well as solute-ligand interactions. Affinity chromatography is an even more versatile method of characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reactants. Adoption of different experimental strategies such as column chromatography, simple partition equilibrium experiments and biosensor technology has created a situation wherein affinity chromatography affords a means of characterizing the whole range of reaction affinities-from relatively weak interactions (binding constants less that 10(3)M (-1)) to tight interactions with binding constants greater than 10(9)M (-1). In addition to its established prowess as a means of solute separation and purification, chromatography thus also possesses considerable potential for investigation of the functional roles of the purified reactants-an endeavour that requires characterization as well as identification of the interactions responsible for a physiological phenomenon.

Analysis of hen egg white lysozyme adsorption on Si(Ti)O 2 ∣ aqueous solution interfaces at low ionic strength: a biphasic reaction related to solution self-association

Adsorption kinetics of hen-egg white lysozyme at concentrations of 10 −7, 10 −6, 10 −4 M and at pH 8.0 have been measured on Si 0.8Ti 0.2O 2 surfaces by means of optical waveguide lightmode spectroscopy and were found to proceed in two distinct kinetic régimes: a fast and quasi linear increase of the surface concentration followed by slower kinetics that could be described qualitatively by the theory of random sequential adsorption. The transition between these two kinetic régimes was correlated with abrupt changes in the values of the mean refractive index and of the thickness of the adsorbed layer as well as in the desorption kinetics performed either before or after the transition from the first régime to the second. Experiments carried out at the same ionic strength but at different pH values showed that the adsorption behaviour of lysozyme is related to its self-association properties in highly concentrated solutions.

Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering

Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation.Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.

NMR detection of oriented association via dilution shifts in tetramethylsilane solvent. 2. Aliphatic esters

Solute interactions are disrupted by the usual NMR solvents. Tetramethylsilane, a moderately good symmetrical non-polar solvent having low polarizability, tends not to interfere with solute self-association. As a result, changes in 1H, 13C (and 19F) deshielding values may be seen upon dilution, and are attributable to dissociation of ‘dimers.’ Solutes show sizable localized dilution shifts near the presumed dimerization site, but near-zero shifts for more distant nuclei, thus providing an ‘intramolecular internal reference.’ Considerable information about the nature and orientation of dimerization, and the weak forces involved, can be derived from routine two-tube NMR data using the measuring capability of modern equipment. The infinite-dilution deshielding values, generated simultaneously, characterize the solute molecule exactly, even in mixtures with other unknown species. Aliphatic esters are found to self-associate by weak non-conventional hydrogen bonds and by polar interactions, by the latter exclusively when no acyl α-hydrogen is present. Copyright © 1999 John Wiley & Sons, Ltd.

Tailoring of specific interactions to modify the morphology of naproxen

Microscopy and spectroscopy were used to deduce the effect of intermolecular hydrogen bonding on the morphology of a recrystallized pharmaceutical compound. Samples of recrystallized naproxen, (+)-6-methoxy- α-methylnaphthaleneacetic acid, were prepared from solutions of benzene, acetone, and methanol resulting in particles with aspect ratios of 2, 3.5, and 5.5, respectively, as determined by SEM imaging. XRDS indicated that the recrystallization did not affect lattice structure. Solubility measurements, UV/Vis, fluorescence, and FTIR spectroscopy were used to investigate specific interactions in solution including solvent–solute hydrogen bonding and solute self-association. The results failed to reveal π orbital conjugation of naproxen even in saturated solutions, but indicated hydrogen bonding of naproxen with the solvent as well as the formation of cyclic naproxen dimers in solution. FTIR microscopy indicated a correlation between solvent and monomeric hydroxyl content in the crystals confirming the effect of solvent on naproxen morphology. The results of this study demonstrate the utility of spectroscopic techniques to tailor specific interactions to modify particle morphology.

Raman Spectroscopic Characterization of Association and Thermoreversible Gelation in Aqueous Systems of Poly(N-acetamidoacrylamide)

Polymer self-association and thermoreversible gelation in aqueous solutions of poly(N-acetamidoacrylamide) (PAAA) have been investigated by Raman spectroscopy. We found that aqueous solutions of PAAA, even at low concentrations, revealed the presence of polymer−polymer coordination, suggesting formation of polymer clusters. The effect of denaturant (sodium thiocyanate) on the inter- and intramolecular interactions in the system has been also studied. It is shown that the SCN ions destroy the macroscopic hydrogen-bond network of water, as well as reduce the polymer's ability for self-association by occupying the NH groups. The changes observed in the Raman spectra upon the gel transition show that in the gel state the equilibrium of the system is shifted toward polymer−polymer coordination, whereas in the solution it is mostly polymer−water coordination. The influence of the different factors such as polymer concentration, level of denaturant addition, and type of solvent on the gel formation is discussed.

Aspects of the molecular structure and dynamics of neuropeptide Y.

Human neuropeptide Y (hNPY) and the Q34-->P34 mutant (P34-hNPY) have been characterized by CD spectroscopy. hNPY self-associates in aqueous solution with a dimerization constant in the micromolar range. The self-association correlates with an increase in secondary-structure content which was studied as a function of concentration, temperature and pH. The effects of temperature were measured in water (5-84 degrees C) and in ethanediol/water (2 : 1) (-90 degrees to +90 degrees C). A single-residue mutation, Q34-->P34, affects the pH, thermal and self-association properties of NPY. The CD results are correlated with photochemically induced dynamic nuclear polarization NMR experiments which show that the tyrosines at the interface between two monomer units present limited accessibility to a photoreactive dye. An equilibrium state is described, involving a PP-fold monomer form and a handshake dimer form, that accommodates the physicochemical properties of NPY.

Effect of Protein Aggregation in the Aqueous Phase on the Binding of Membrane Proteins to Membranes

Analysis of the binding of hydrophobic peptides or proteins to membranes generally assumes that the solute is monomeric in both the aqueous phase and the membrane. Simulations were performed to examine the effect of solute self-association in the aqueous phase on the binding of monomeric solute to lipid vesicles. Aggregation lowered the initial concentration of monomeric solute, which was then maintained at a relatively constant value at the expense of the aggregated solute, as the lipid concentration was increased. The resultant binding isotherm has a more linear initial portion rather than the classic hyperbolic shape. Although this shape is diagnostic of solute self-association in the aqueous phase, various combinations of values for the membrane partition coefficient and the solute self-association constant will generate similar isotherms. Data for cytochrome b 5 were analyzed and, when the self-association constant was estimated by gel filtration, a unique value for the membrane partition coefficient was obtained. Thus, to obtain a true partition coefficient the state of the solute in the aqueous phase must be known. If the concentration of the monomeric solute species in the aqueous phase can be independently determined, then, even with heterogeneous aggregates, the true partition coefficient can be obtained.

Synthesis and Self-Association of 4-Pyrimidinones

Crystallization of 4-pyrimidinone from CHCl3 produced colorless needles that were shown by X-ray crystallography to consist of cyclic hydrogen-bonded dimers 2 of the 4(3H)-pyrimidinone tautomer 1. Vapor-pressure osmometry established that a simple derivative, 5-(2-phenylethyl)-4(3H)-pyrimidinone (5), self-associates in solution with a value of Ka (220 ± 110 m-1 at 26 °C in CH2Cl2) close to that measured for 2-pyridinone under similar conditions. These observations suggest that 4-pyrimidinones and 2-pyridinones have similar modes and degrees of association and should be equally suitable for use as sticky functional groups that can be incorporated in complex molecules to make them associate in particular ways. This hypothesis was tested by synthesizing three dipyrimidinones (12, 16, and 21) designed to form strongly hydrogen-bonded dimers and by making a tetrapyrimidinone (26) designed to self-associate and to thereby generate a three-dimensional hydrogen-bonded network. In all cases, however, the compounds...

Study of self-association of 2-methyl-2-butanol based on non-linear dielectric effect

Non-linear electrical polarisation was applied to studies of self-association of solutions of 2-methyl-2-butanol in cyclohexane in the whole concentration range at temperatures from 287 to 318 K. The free energy, enthalpy and entropy of hydrogen bonds in chain multimers and low polar cyclic tetramers were determined. The angle β between dipole moments of two neighbouring molecules was found to be close to tetrahedral. The mean association number ( χ) amounts to 2.5 for pure alcohol. The fraction of cyclic species rapidly increases in diluted solutions, reaching almost a constant value of 80% for concentrated solutions and pure alcohol.

Structural and functional consequences of an N-glycosylation mutation (HEMPAS) affecting human erythrocyte membrane glycoproteins.

Band 3, the human erythrocyte anion exchanger (AE1), and the glucose transporter (GLUT1) proteins each contain a single site of N-glycosylation that is heterogeneously glycosylated. Lectin binding and enzymatic deglycosylation assays showed that the polylactosaminyl oligosaccharide structure of these glycoproteins was altered to a high mannose or hybrid glycan form in three patients with hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS). Offspring from one of the HEMPAS patients had intermediate levels of polylactosaminyl oligosaccharide associated with AE1 and GLUT1, suggesting they may have been heterozygous for the genetic defect. The array of polylactosaminyl-containing glycoproteins present in EBV-transformed lymphoblasts derived from fresh blood of HEMPAS patients was similar to control lymphoblasts. HEMPAS lymphoblasts do not therefore express the defect in polylactosamine synthesis found in erythroid cells, indicating that lymphoid cells are not deficient in the processing enzymes or contain an alternative oligosaccharide processing pathway. Purified HEMPAS band 3 had an unaltered oligomeric structure but dimers aggregated more rapidly in detergent solution than normal band 3. The altered oligosaccharide structure did not affect the sensitivity of band 3 to proteolytic digestion in intact red cells but a greater amount of HEMPAS band 3 was associated with the cytoskeleton. The transport activities of AE1 and GLUT1 in HEMPAS erythrocytes were similar to those in normal controls. This shows that the HEMPAS glycosylation defect does not impair the functional accumulation of these two important erythrocyte membrane transporters even though it produces subtle structural changes in band 3 that result in its increased cytoskeletal interaction and self association in detergent solution.

Self-association of new mixed-ligand diimine–N-acyl-N′,N′-dialkyl thioureate complexes of platinum(II) in acetonitrile solution †

The complexes [Pt(bipy)L]PF6 and [Pt(phen)L]PF6 (HL = N-acyl-N′,N′-di-n-butylthiourea) showed pronounced self-association in CD3CN solution at 25 °C according to an equilibrium 2[Pt(diimine)(L-S,O)]+ ⇌ [{Pt(diimine)(L-S,O)}2]2+ for which association constants, KD, have been estimated from the concentration dependence of the 1H NMR shifts of these complexes. The values of KD range from 1.8 to 114 M–1 depending on the structure of the diimine as well as the nature of the N-acyl group; the values of the 1,10-phenanthroline complexes are generally larger than those for the corresponding 2,2′-bipyridine analogues. In general, N-1-naphthoyl-N′,N′-dibutylthiourea complexes self-associate to a lesser extent than the corresponding N-benzoyl analogues. From the temperature dependence of the KD values, the thermodynamic parameters ΔG, ΔH and ΔS have been estimated. An average Δ(ΔG) increment of 2.4 ± 0.4 kJ mol–1 per aromatic ring of the diimine moiety co-ordinated to the complexes is consistent with energies generally associated with π–π stacking and cation π interactions observed for other systems.

Insulin lispro: a review of its pharmacological properties and therapeutic use in the management of diabetes mellitus.

Insulin lispro, a recombinant insulin analogue, is identical to human insulin except for the transposition of proline and lysine at positions 28 and 29 in the C-terminus of the B chain. The resultant reduced capacity for self-association in solution translates into more rapid absorption of insulin lispro than human regular insulin from subcutaneous sites. Maximum insulin concentrations are higher and are reached earlier with insulin lispro than with human regular insulin, and insulin concentrations return to baseline values more quickly with insulin lispro; consequently, insulin lispro has a more rapid onset and a shorter duration of glucose-lowering activity. These pharmacological properties provided the rationale for comparative clinical trials of subcutaneous insulin lispro (administered within 15 minutes before meals, preferably immediately before meals) and subcutaneous human regular insulin (administered 20 to 45 minutes before meals) in patients with type 1 diabetes (insulin-dependent diabetes mellitus) or type 2 diabetes (non-insulin-dependent diabetes mellitus) requiring premeal insulin therapy plus basal insulin therapy. Available clinical trials are well designed and results suggest that 1- and 2-hour postprandial blood glucose levels with insulin lispro are similar to or lower than those with human regular insulin; 1- and 2-hourpostprandial glucose excursions are similar to or less pronounced than those with human regular insulin. Glycated haemoglobin A values were generally similar with both agents. Continuous subcutaneous insulin infusion was associated with greater improvements in postprandial blood glucose levels and glycated haemoglobin A1 values with insulin lispro than with human regular insulin. Confirmatory data are required. The incidence of hypoglycaemia with insulin lispro was similar to or lower than that with human regular insulin. In particular insulin lispro appears to be associated with a lower incidence of night-time and severe hypoglycaemic episodes. Evidence also suggests that patients perceive their quality of life to be improved with insulin lispro compared with human regular insulin, and that satisfaction with treatment is greater with the insulin analogue. Thus, in patients with type 1 or 2 diabetes requiring premeal insulin therapy, insulin lispro appears to provide greater postprandial glycaemic control than human regular insulin without increasing the risk of hypoglycaemia. Furthermore, the reduced injection-meal interval with this agent offers greater convenience for the patient than regular human insulin. If longer term clinical experience supports these promising results it is likely that insulin lispro will offer important advantages over human regular insulin.

Low temperature solution behaviour of Methylophilus methylotrophus electron transferring flavoprotein: a study by analytical ultracentrifugation

The solution behaviour of electron transferring flavoprotein (ETF) from Methylophilus methylotrophus was investigated at low temperature (4 °C) by analytical ultracentrifugation. The concentration dependence of the apparent weight average molecular weight, Mw,app, established the existence of the protein in heterodimeric state (M = 63,700 Da), but also signified the possible dissociation of the heterodimer at lower concentrations into its constituent subunits (M = 28,900 Da and 33,700 Da, together with FAD and AMP cofactors of collective M = 1120 Da). This similarity in subunit size allows approximate quantification of the dissociation in terms of expressions for a monomer-dimer equilibrium. The dissociative behaviour was confirmed by determination of the point average molecular weight, Mw,app(r), as a function of the ETF concentration, c(r), throughout the sedimentation equilibrium distributions obtained with loading concentrations of 0.4 and 0.7 mg/ml. By means of the recently formulated ``psi'' procedure for direct analysis of solute self-association a value of (1.5 ± 0.1) µM has been obtained for the dissociation constant Kd. Sedimentation velocity experiments yielded an estimate of the heterodimer sedimentation coefficient, s020,w, of (4.5 ± 0.2) S which for M = 63,700 Da suggests a globular structure.