Self-association In Solution Research Articles

Allowance for the effect of protein charge in the characterization of nonideal solute self-association by sedimentation equilibrium

This theoretical investigation explores the use of statistical–mechanical approaches to characterize the reversible tetramerization of a protein monomer with the size and charge characteristics of serum albumin under conditions where consideration of nearest-neighbor interactions suffices to describe effects of thermodynamic non-ideality. Such analysis of simulated sedimentation equilibrium distributions points to the adequacy of both the scaled particle theory and potential-of-mean-force methods for determining the self-association constant. Although the latter method usually entails the assignment of a magnitude to monomer net charge, this requirement can be obviated to some extent by repeating the analysis for a range of monomer charges and identifying the most appropriate value as that associated with a minimum in the sum-of-squares-of-residuals (SSR) of the best-fit descriptions of the sedimentation equilibrium distribution. Reasonable estimates of the association constant are usually obtained from corresponding analyses of the same sedimentation equilibrium distributions with activity coefficients obtained by scaled particle theory, an approach which also involves the identification of parameters on the basis of a minimum in SSR. However, the value of monomer charge determined must be regarded as a curve-fitting parameter rather than a true measure of monomer charge. Similar qualifications are shown to prevail in the scaled particle theory approach, which also involves the identification of parameters (the effective monomer volume and the polymer/monomer volume ratio) on the basis of a minimum in SSR. We therefore recommend discontinuation of the practice whereby quite precise distinction between modes of self-association has been attempted on the grounds of the physical credibility of the magnitudes of these additional curve-fitting parameters.

Evidence for Self-Association of Nonionic and Other Organic Solutes in Liquid Phases Comprising 1-Octanol and Water

We use a monomer-single-multimer model to judge whether there is significant self-association of an organic solute distributed between 1-octanol and water. Self-association leads to octanol-water partition coefficients, K(ow), that depend upon the concentration of solute and this affects their application, significantly so for lipophilic compounds. Our measurements, done over as wide a range of concentration as possible, suggest that: (1) For toluene, there is dimerization in the water and tetramerization in 1-octanol. (2) For p-xylene, there is significant self-association of unknown degree in 1-octanol. (3) Biphenyl exhibits no self-association in either phase. The model confirms the conclusion that there is self-association of nicotimamide only in the aqueous phase, a conclusion reached in original measurements and interpretation (Charman, W. N.; Lai, C. S. C.; Finnin, B. C.; Reed, B. L. Pharm. Res. 1991, 8, 1144-1150). Our analysis of published measurements on the four isomers of hexachlorocyclohexanes (Paschke, A.; Shuurmann, G. Chem. Eng. Technol. 2000, 23, 666-670) leads to the conclusion that there is significant self-association of unknown degree in the aqueous phase. There is a discernible region of concentration-independent behavior as infinite dilution is approached in the aqueous phase, except notably for the hexachlorocyclohexanes. We suggest this is due to self-association incorporating the solvent to form multimer-solvent complexes. The data suggest that self-association, when it occurs, has a greater significance in the more lipophilic cases and this may partly explain why the variability in measurements of octanol-water partition coefficients between laboratories tends to be larger and significant for more lipophilic solutes.

Influence of Conformation of 9,10-Anthrylene Rotors on Structures and Self-association Properties of Macrocyclic Arylene–Alkynylene Oligomers

Abstract Macrocyclization of 9,10-bis[(3-ethynyl-5-octylphenyl)ethynyl]anthracene afforded three cyclic oligomers: a dimer, a trimer, and a tetramer. Only the trimer showed significant self-association in solution because of its fully planar structure. Their structures and properties were significantly influenced by the conformation of the 9,10-anthrylene rotors depending on the ring size.

Self-Assembling Thiophene Dendrimers with a Hexa-peri-hexabenzocoronene Core−Synthesis, Characterization and Performance in Bulk Heterojunction Solar Cells

The solid state organization of molecules is an important factor in determining the performance of organic electronic devices. In bulk heterojunction (BHJ) solar cells, the arrangement of electron donor and acceptor materials into distinct crystalline phases of ideal size and distribution can lead to better power conversion efficiencies. The use of fluorenyl hexa-peri-hexabenzocoronene (FHBC) 2 in this study has highlighted the importance of molecular organization to device performance. FHBC compounds 6, 8, and 10, functionalized with a series of thiophene dendrons, were synthesized using Suzuki−Miyaura coupling in high yields. In UV−vis and 1H NMR spectroscopic studies, all FHBC derivatives showed self-association in solution. Hexagonal packing of columnar structures was observed for solid state samples of FHBC 2 and 8 in two-dimensional wide-angle X-ray scattering experiments. In thin film X-ray experiments, ordered structures were observed in blends of FHBC 2 and fullerene acceptor materials indicating that there is phase separation between the donor and acceptor materials and that the self-organization of the FHBC material is unaffected. While the large thiophene dendritic substituent attached to compound 10 broadened its UV−vis absorption profile, the solid state morphology is altered by the bulky thiophene dendrons. These molecular structure variations are reflected in the performance characteristics of BHJ solar cell devices fabricated using these FHBC compounds as electron donor materials. Power conversion efficiency of 2.5% was achieved for a device containing compound 10 with [6,6]-phenyl-C71-butyric acid methyl ester (PC71BM) as the acceptor material. This compares favorably with devices fabricated with pure dendritic thiophene materials and illustrates the positive effect of molecular self-organization on device performance.

Developing force fields from the microscopic structure of solutions

We have been developing force fields designed for the eventual simulation of peptides and proteins using the Kirkwood–Buff (KB) theory of solutions as a guide. KB theory provides exact information on the relative distributions for each species present in solution. This information can also be obtained from computer simulations. Hence, one can use KB theory to help test and modify the parameters commonly used in biomolecular studies. A series of small molecule force fields representative of the fragments found in peptides and proteins have been developed. Since this approach is guided by the KB theory, our results provide a reasonable balance in the interactions between self-association of solutes and solute solvation. Here, we present our progress to date. In addition, our investigations have provided a wealth of data concerning the properties of solution mixtures, which is also summarized. Specific examples of the properties of aromatic (benzene, phenol, p-cresol) and sulfur compounds (methanethiol, dimethylsulfide, dimethyldisulfide) and their mixtures with methanol or toluene are provided as an illustration of this kind of approach.

Comparison of Methods for Characterizing Nonideal Solute Self-Association by Sedimentation Equilibrium

We have examined in detail analytical solutions of expressions for sedimentation equilibrium in the analytical ultracentrifuge to describe self-association under nonideal conditions. We find that those containing the radial dependence of total solute concentration that incorporate the Adams-Fujita assumption for composition-dependence of activity coefficients reveal potential shortcomings for characterizing such systems. Similar deficiencies are shown in the use of the NONLIN software incorporating the same assumption about the interrelationship between activity coefficients for monomer and polymer species. These difficulties can be overcome by iterative analyses incorporating expressions for the composition-dependence of activity coefficients predicted by excluded volume considerations. A recommendation is therefore made for the replacement of current software packages by programs that incorporate rigorous statistical-mechanical allowance for thermodynamic nonideality in sedimentation equilibrium distributions reflecting solute self-association.

STUDIES ON SELF-ASSOCIATIVE BEHAVIOR OF A NOVEL CATION AMPHIPHILIC POLYMER

A novel associating polymer P(AEBA) was synthesized by radical polymerization of the cationic amphiphilic monomer, 4-(2-(acryloyloxy)ethoxy)benzyl tri-ethyl ammonium bromide (AEBA), in aqueous solutions. P(AEBA) displays a strong tendency for self-association in aqueous solutions and is sensitive to the external stimulation such as added salt. The associative properties and morphologies of P(AEBA) were studied by fluorescnece probe technique, viscometry and TEM. In dilute salt-free solutions P(AEBA) behaves as polyelectrolyte, while its behavior is similar to that of the polysoap as salt added. Accompanying increasing polymer concentration, polymer aggregation conformation changes from an extended necklace-like structure to a compact globular aggregate corresponding to the viscosity reduction.

Glycine Exists Mainly as Monomers, Not Dimers, in Supersaturated Aqueous Solutions: Implications for Understanding Its Crystallization and Polymorphism

Glycine, the simplest amino acid, is described as existing as hydrogen-bonded cyclic dimers in supersaturated aqueous solutions and, as a result, crystallizing in a centrosymmetric polymorph (polymorph alpha) for which the dimer can be viewed as the building unit, in favor of other polymorphs of polar structures. In exhibiting this relation between polymorphic selectivity and self-association in solution, glycine is thought to illustrate a general principle. We measured the freezing-point depression of glycine-water up to 30% supersaturation and found that glycine exists mainly as monomers, not dimers, and that the dimer stability constant K D is smaller than 0.1 kg of H 2O/mol if the observed osmotic abnormality is attributed to dimerization. We also revisited a report cited as evidence for glycine dimerization: the slowdown of glycine diffusion with solution age. Pulsed gradient spin-echo NMR spectroscopy was used in place of the previous method of Gouy interferometry to avoid perturbations to sloution structure caused by the interdiffusion between two solutions of different concentrations. No aging effect was observed on glycine diffusion up to 24% supersaturation after five days. The solute size calculated from diffusivity, viscosity, and the Stokes-Einstein relation showed no increase with concentration or solution age. We conclude that glycine exists in supersaturated aqueous solutions mainly as monomers, not dimers, and remains so upon aging. This result does not invalidate the theories of how pH and additives affect glycine's polymorphic preference, because they begin with the assumption that alpha glycine is the preferred polymorph under normal conditions, but requires a new explanation for that assumption itself.

Experimental Dipole Moments for Nonisolatable Acetic Acid Structures in a Nonpolar Medium. A Combined Spectroscopic, Dielectric, and DFT Study for Self-Association in Solution

Acetic acid can exist in many possible structural forms depending on its surrounding medium. A recently developed inverse problem methodology (J. Phys. Chem. B 2007, 111, 13064-13074) was utilized in order to elucidate acetic acid structures in a dilute nonpolar medium. In this regard, simultaneous and stopped-flow measurements of the bulk solution densities, refractive indices, relative permittivities, and IR spectra of acetic acid in toluene were performed at several different concentrations in a semibatch closed-loop experimental setup at 298.15 K and 0.1013 MPa. This combined IR spectroscopic and dielectric, density, and refractive index analysis was employed in order to distinguish acetic acid structures and to further determine the dipole moments of the monomer, cyclic dimer, and "lumped-sum" open dimers. The infrared spectra were first analyzed to provide qualitative understanding as well as quantitative estimates for each acetic acid species. Subsequently, the dipole moments of these species were calculated using a direct approach which was primarily based on response surface models. The present method allows the determination of individual dipole moments not only for the monomer but also for the cyclic dimer and the open dimer. The results obtained from this study experimentally show that the cyclic dimer with centrosymmetric structure has a dipole moment approximately 0 D. The results also suggest that the linear dimers are present as mixtures of linear dimers structures. The existence of the linear dimers mixture was also indicated by the experimental infrared analysis of the OH-stretching region (particularly for measurements in n-hexane as solvent) and comparison of these spectra with DFT predictions. Finally, the present methodology which incorporates simultaneous physicochemical and spectroscopic analysis is undoubtedly useful for physicochemical characterization for other nonisolatable solute species and self-associated structures in solution.

Visualization of Transient Ultra-Weak Protein Self-Association in Solution Using Paramagnetic Relaxation Enhancement

Ultra-weak macromolecular self-association is exceptionally difficult to both detect and visualize using conventional biophysical techniques owing to the very low population of the associated species, yet such weak intermolecular interactions coupled with nucleation events play an important role in driving spontaneous self-assembly to form higher-order architectures (such as crystals, viral capsids, and amyloid fibrils). In this article, we detect and characterize transient, ultra-weak self-association (KD >or= 15 mM) involving the histidine-containing protein HPr by means of paramagnetic relaxation enhancement (PRE), using EDTA-Mn2+ conjugated at three separate sites (E5C, E25C, and E32C, one at a time). Large intermolecular PRE effects, above the background observed with hydroxylamine-EDTA-Mn2+ as a control, are observed for two of the three paramagnetically labeled sites, E5C and E32C. The extent of self-association can be modulated (significantly reduced) by increasing the ionic strength or by the introduction of a negative charge (S46D mutation) within a positively charged surface patch, and abolished upon the addition of the N-terminal domain of enzyme I (EIN) to form a specific EIN-HPr complex. The PRE profiles observed for E5C and E32C can be fitted simultaneously and accounted for quantitatively using conjoined rigid body/torsion angle dynamics-simulated annealing with an ensemble of states to represent the distribution of one molecule of HPr relative to its partner.

Structure−Property Relationships of Fluorinated Donor/Acceptor Tetrakis(phenylethynyl)benzenes and Bis(dehydrobenzoannuleno)benzenes

Nine new bisdonor/bisacceptor-functionalized tetrakis(phenylethynyl)benzene (TPEB) and six new bis(dehydrobenzoannuleno)benzene (DBA) chromophores have been synthesized. The compounds consist of electron-donating dibutylaniline groups connected through a conjugated phenyl-acetylene scaffold to benzotrifluoride, bis(trifluoromethyl)phenyl, or pentafluorophenyl acceptor groups. In comparison to previously reported analogues utilizing nitrophenyl or benzonitrile acceptor groups, the weaker acceptor groups exhibit visibly fluorescent intramolecular charge transfer (ICT) behavior, moderately narrow optical band gaps, moderately high quantum yields, and strong fluorescence solvatochromism. In this series of molecules, the strongly inductive fluoro acceptor groups result in optical properties similar to the resonance acceptor analogues, making them promising candidates for optical materials device components. The data also support recent investigations that question the utility of using UV/vis spectroscopy alone as a qualitative measure of conjugation. The bisDBAs exhibit weaker ICT behavior and self-association in solution than their corresponding nitro analogues, but show greater stability to decomposition via polymerization and smaller optical band gaps than their acyclic analogues.

Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies.

Cardiac Troponin T Forms a Tetramer in Vitro

Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction has led to the development of antibody-based assays for measuring cTnT serum concentration. We examined the behavior of cTnT in solution. Recombinant human cTnT3, the dominant isoform in the adult human heart, was used. The protein was pure and functional, as demonstrated by SDS-PAGE and surface plasmon resonance. cTnT3 was found to bind specifically and in a concentration-dependent manner to cTnC. Routine size exclusion chromatography suggested a higher-than-expected MW for cTnT. Using analytical ultracentrifugation, we found cTnT3 in solution to be mainly in the form of a tightly bound tetramer at concentrations as low as 4 micromol/L. Our sedimentation velocity and transmission electron microscopy results indicate that the tetramer's shape is elongated rather than globular. CTnT's self-association in solution is an important consideration in the design and interpretation of experiments with the aim of understanding the biochemical and biophysical properties of cTnT, its isoforms, and its mutants.

Clinical Pharmacokinetics and Pharmacodynamics of Insulin Glulisine

Insulin glulisine injection [3(B)-Lys, 29(B)-Glu-human insulin] is the newest human insulin analogue product for the control of mealtime blood sugar. As with insulin aspart and insulin lispro products, the insulin glulisine product displays faster absorption and onset of action, with a shorter duration of action than that of regular human insulin. The modifications of the amino acid sequence at positions 3 and 29 in the B chain of human insulin simultaneously provide stability to the molecular structure and render the insulin glulisine molecule less likely to self-associate, compared with human insulin, while still allowing the formation of dimers at pharmaceutical concentrations. Unlike other insulin analogue products, this allows for a viable drug product in the absence of hexamer-promoting zinc and, thus, provides immediate availability of insulin glulisine molecules at the injection site for absorption. Pharmacokinetic studies with insulin glulisine have shown an absorption profile with a peak insulin concentration approximately twice that of regular human insulin, which is reached in approximately half the time. Dose proportionality in early, maximum and total exposure is observed for insulin glulisine over the therapeutic relevant dose range up to 0.4 U/kg. The pharmacodynamic profile of insulin glulisine reflects the absorption kinetics by demonstrating a greater rate of glucose utilization, which is completed earlier and at equipotency on a molar base compared with regular human insulin. Dose-proportionality in glucose utilization has been established for insulin glulisine in patients with type 1 diabetes mellitus in the dose range of 0.075-0.15 U/kg, and a less than dose-proportional increase above 0.15 U/kg, indicating saturation of insulin action in general. The rapid absorption and action of insulin glulisine show similar low intrasubject variability compared with insulin lispro and regular human insulin when given repeatedly, and have been confirmed in healthy subjects of different body mass indices (BMIs) and ethnic groups, as well as adults and children with type 1 and type 2 diabetes. Furthermore, the early insulin exposure and action of insulin glulisine were slightly -- but consistently -- greater than those of insulin lispro in healthy volunteers across a wide range of BMIs.Meal studies in patients with type 1 diabetes show that insulin glulisine provides better postprandial blood glucose control than regular human insulin when administered immediately pre-meal, and equivalent control when given after the meal. In a study in patients with type 2 diabetes, the overall postprandial blood glucose excursions were lower with insulin glulisine than with insulin lispro. Therefore, by virtue of its primary structure, insulin glulisine demonstrates both low self-association in solution and stability for a viable insulin product in the absence of zinc, thereby maintaining immediate availability for absorption after subcutaneous injection. This confers the most rapid onset of glucose-lowering activity and adds to the flexibility in postprandial blood glucose control.

Physicochemical Studies of Octadecyltrimethylammonium Bromide: A Critical Assessment of Its Solution Behavior with Reference to Formation of Micelle, and Microemulsion with n-Butanol and n-Heptane

Octadecyl-trimethyl-ammonium bromide (C18TAB) is a much less studied representative in the alkyltrimethylammonium halide surfactant series. A comprehensive study of its normal and reverse micelle (microemulsion) formation has been herein conducted by the methods of conductometry, tensiometry, fluorimetry, and microcalorimetry. The energetics of its air/liquid interfacial adsorption and self-association in aqueous solution have been examined. The phase behavior of its combinations with water, n-butanol, and n-heptane in the formation of microemulsions have been investigated with identification of a variety of phases. The energetics of formation of water dispersion in oil (w/o) has been evaluated from dilution experiments conducted at different temperatures. From the results, structural parameters of the droplets have been determined at different [water]/[surfactant] mole ratios (omega) and temperatures. The w/o dispersions have evidenced both volume- and temperature-induced conductance percolation. The results have been treated in light of the Scaling equations, and the associated parameters for the process have been determined. The activation energies for the temperature-induced percolation process of the w/o dispersion have been evaluated and assessed.

Pyrene Derivatives of 2,2′-Bipyridine as Models for Asphaltenes: Synthesis, Characterization, and Supramolecular Organization

The behavior of 4,4′-bis-(2-pyren-1-yl-ethyl)-[2,2′]bipyridinyl (PBP) was studied as a model for petroleum asphaltenes with a bridged structure. PBP consists of two pyrene groups bridged by a bipyridyl spacer, and exhibits similar solubility and chromatographic properties to some fractions of asphaltenes. On the basis of nuclear magnetic resonance, steady state fluorescence, vapor pressure osmometry, solubility, and adsorption behavior studies, PBP gave self-association in solution. On the basis of these data and single crystal X-ray analysis, this behavior was attributed to π–π stacking interactions involving both pyrene rings and the bipyridine spacer. These results demonstrate that bridged aromatic species with up to four fused aromatic rings are capable of self-association in solution.

Self-association of adenine-dependent hairpin ribozymes

Hairpin ribozymes are flexible molecules that catalyse reversible self-cleavage after the docking of two independently folded internal loops, A and B. The activities, self-association and structures in solution of two 85 base adenine-dependent hairpin ribozymes (ADHR1 and ADHR2) were studied by native gel electrophoresis, analytical centrifugation, and small angle neutron scattering. Bi-molecular RNA interactions such as linear-linear, loop-loop, loop-linear or kissing interactions have been found to be important in the control of various biological functions, and hairpin loops present rich potential for establishing both intra- and intermolecular interactions through standard Watson-Crick base pairing or non-canonical interactions. Similar results were obtained for ADHR1 and ADHR2. At room temperature, they indicated end-to-end self-association of the ribozymes in rod-like structures with a cross-section corresponding to two double strands side-by-side. Dimers, which predominate at low concentration ( approximately 0.1 mg/ml), associate into longer rods, with increasing concentration ( approximately 1 mg/ml). Above 65 degrees C, the dimers and rods dissociated into compact monomers, with a radius of gyration similar to that of tRNA (about 70 bases). The dimers were non-active for catalysis, which suggests that dimer formation, probably by preventing the correct docking of loops A and B, could act as an inhibition mechanism for the regulation of hairpin ribozyme catalysis.

TPR-Mediated Self-Association of Plant SGT1

The tetratricopeptide repeat (TPR) domain mediates inter-protein associations in a number of systems. The domain is also thought to mediate oligomerization of some proteins, but this has remained controversial, with conflicting data appearing in the literature. By way of investigating such TPR-mediated self-associations we used a variety of biophysical techniques to characterize purified recombinant Sgt1, a TPR-containing protein found in all eukaryotes that is involved in a broad range of biological processes, including kinetochore assembly in humans and yeast and disease resistance in plants. We show that recombinant Sgt1 from Arabidopsis, barley, and yeast self-associates in vitro while recombinant human Sgt1 does not. Further experiments on barley Sgt1 demonstrate unambiguously a TPR-mediated dimerization, which is concentration- and ionic-strength-dependent and results in a global increase in helical structure and stability of the protein. Dimerization is also redox sensitive, being completely abolished by the formation of an intramolecular disulfide bond where the contributing cysteines are conserved in plant Sgt1s. The dimer interface was mapped through cross-linking and mass spectrometry to the C-terminal region of the TPR domain. Our study, which provides the first biophysical characterization of plant Sgt1, highlights how TPR domains can mediate self-association in solution and that sequence variation in the regions involved in oligomerization affects the propensity of TPR-containing proteins to dimerize.

Relationship between Self-Association of Glycine Molecules in Supersaturated Solutions and Solid State Outcome

Small angle x-ray scattering is utilized to directly examine the formation of clusters in supersaturated solutions of glycine, in an attempt to understand their role in the nucleation process. The results suggest that the majority of glycine molecules exist as dimers in aqueous solutions, and monomers in 13% (upsilon/upsilon) acetic acid-water mixtures. As the water and acetic acid-water solutions crystallize into alpha and gamma forms, respectively, the findings indicate a direct correlation between molecular self-association in solution and the polymorphic outcome.

Boron Dipyrromethene Dyes Bearing Ancillary 2,2′:6′,2″‐Terpyridine Coordination Sites

AbstractTwo new boron‐dipyrromethene dyes have been synthesised and their photophysical properties have been investigated by steady‐state and time‐resolved fluorescence spectroscopy. These dyes are equipped with ancillary 2,2′:6′,2″‐terpyridine (terpy) residues, attached either at the B centre or at the meso position of the organic framework; in this latter case two ethynylated pyrene units are bound to the B atom. Under most experimental conditions, the absorption and emission spectral properties are typical of this class of dye. Complexation of zinc(II) cations to the terpy units attached at the B centre causes complete extinction of the fluorescence due to intramolecular electron transfer from the excited state dye to the resultant zinc(II) bis‐terpy complex. Similar behaviour is noted when the terpy is attached at the meso site, although the rate of electron transfer is slower due to the weaker driving force. A most unusual feature of the pyrene‐substituted dye is its facile self association in acetonitrile solution. This results in the reversible formation of a fluorescent associate, believed to be a symmetric dimer, that absorbs and emits to the red of the monomer. Self association is considered to be driven by a solvophobic effect in which the pyrene units seek to minimize surface contact with the surrounding solvent molecules.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)