Statistical thermodynamics of nucleic acid melting transitions with coupled binding equilibria

Equations are developed to describe the shift in the temperature of the helix–coil transition when small molecules bind to nucleic acids. Included are high polymers, oligonucleotides, and oligomer–polymer interactions. The equations prescribe simple ways of plotting experimental data to evaluate transition and binding parameters.     

Biological and protein‐binding studies of newly synthesized polymer–cobalt(III) complexes

The polymer–cobalt(III) complexes, [Co(bpy)(dien)BPEI]Cl₃ · 4H₂O (bpy = 2,2′‐bipyridine, dien = diethylentriamine, BPEI = branched polyethyleneimine) were synthesized and characterized. The interaction of these complexes with human serum albumin (HSA) and bovine serum albumin (BSA) was investigated under physiological conditions using various physico‐chemical techniques. The results reveal that the fluorescence quenching of serum albumins by polymer–cobalt(III) complexes took place through static quenching. The binding of these complexes changed the molecular conformation of the protein considerably. The polymer–cobalt(III) complex with x = 0.365 shows antimicrobial activity against several human pathogens. This complex also induces cytotoxicity against MCF‐7 through apoptotic induction. However, further studies are needed to decipher the molecular mode of action of polymer–cobalt(III) complex and for its possible utilization in anticancer therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol–water solutions

Highly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, L₀, typically = 12–15 cm) for systematic measurements of the fiber length L in ethanol–water solutions, using a simple mechanochemical set up. The decrease in relative length L/L₀ with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% (v/v) ethanol for NaDNA fibers and at 80 and 84% ethanol for K- and CsDNA fibers. A smaller decrease in L/L₀ of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol–water solutions, the heat-induced helix–coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T_m, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na- and KDNA fibers showed a local maximum in T_m. On further increase of the ethanol concentration, the A-DXA region followed with an even steeper linear decrease in T_m. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na- and KDNA the T_m dependence in and around the A-B transition region could be expressed as a weighted mean value of T_m of A- and B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T_m increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments (～ 80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion–water affinity model. In another series of measurements, T_m of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported by calculations within the Poisson–Boltzmann cylindrical cell model. © 1994 John Wiley & Sons, Inc.     

Helix–random chain transitions of polyamino acids in organic acid–salt solutions

Polyamino acids which are soluble and helical in acetic acid and dichloroacetic acid (DCA) have been observed to undergo a helix to random chain transition upon the addition of lithium salts of strong acids. The transition can be reversed by diluting the salt. Apparently only lithium cations are able to bring about the polycarbobenzoxy-L -lysine (PCBL) transition in acetic acid, whereas the anions display a varying degree of effectiveness; ClO₄^? > Br^? > TSA^? > Cl^? > NO₃^?. The lithium salts of carboxylate anions such as OAc^? and TFA^? do not cause polymer unwinding in acetic acid. Neither do the acids, TSA, HCl, TFA, or DCA induce the transformation in acetic acid. Poly-L -alanine (PLA) in DCA unfolds as LiBr is added, but does not unfold in the presence of 0.5M (CH₃)₄NBr, 0.25M CsBr, or 0.32M HCl. These results are explained on the basis of a direct interaction of the lithium salt with the polymer amide groups to form an ion-pair complex. The extent to which the union of the ion pair can dissociate from the complex in the low dielectric constant, environment determines the degree of unfolding of the polymer. The anion dissociation equilibrium presumably therefore would lie in the same order as given above. Acids such as HCl and TSA are considered to substantially protonate and ion-pair with the polymer, but do not readily dissociate the anion partner from the complex, and therefore do not produce an unstable positively charged helical structure.     

Helix–random coil phase transitions in polypeptide systems with random defects: A statistical mechanical model

The statistical mechanics of the effect of a small number of randomly occurring defects on the melting of a polypeptide chain is developed by using a perturbation of Boltzmann factors technique. The method is illustrated by an example where a second-component impurity contributes more to the energy of the hydrogen-bonded superstructure of the polypeptide chain than does the dominant amino acid residue, resulting in the overall destabilization of the helical conformation.     

Helix–coil transitions of compositionally heterogeneous DNA

Fragmented and mitomycin C cross-linked E. coli DNA was fractionated according to base composition by means of hydroxylapatite chromatography and density-gradient centrifugation in order to determine the effect of compositional heterogeneity on the breadth of the helix–coil transition. The transitions of some of the fractions are broader than that of the unfractionated DNA, due, presumably, to nonrandom sequences in molecules of 5 × 10⁵ daltons. Analysis of the transition breadths in terms of the known heterogeneity leads to reconsideration of current DNA helix–coil transition theory. We propose that partially denatured states include those for which the chains do not remain in strict register. Denaturation profiles are comprehensible only if this multitude of entropically favorable, degenerate states is included in the theory.     

Phase transitions of the starch–water system

When purified potato starch granules are heated in the presence of limited amounts of water (less than 1.5 H₂O: starch, w/w), two endothermic transitions are observed by differential scanning calorimetry. The lower temperature endotherm is always observed at a fixed temperature, 66°C; it is the only endotherm observed when excess water is present. The higher temperature endotherm is observed at increasing temperatures as the water content is decreased. The size of this endotherm decreases with water content. The appearance of the higher temperature endotherm allows the determination of the stoichiometry for full hydration of starch, 14 H₂O/hexose unit. The shift of the higher temperature endotherm is interpreted as the lowering of the melting point of starch crystallites by solvent water.     

Sterol structure determines miscibility versus melting transitions in lipid vesicles

Lipid bilayer membranes composed of DOPC, DPPC, and a series of sterols demix into coexisting liquid phases below a miscibility transition temperature. We use fluorescence microscopy to directly observe phase transitions in vesicles of 1:1:1 DOPC/DPPC/sterol within giant unilamellar vesicles. We show that vesicles containing the "promoter" sterols cholesterol, ergosterol, 25-hydroxycholesterol, epicholesterol, or dihydrocholesterol demix into coexisting liquid phases as temperature is lowered through the miscibility transition. In contrast, vesicles containing the "inhibitor" sterols androstenolone, coprostanol, cholestenone, or cholestane form coexisting gel (solid) and liquid phases. Vesicles containing lanosterol, a sterol found in the cholesterol and ergosterol synthesis pathways, do not exhibit coexisting phases over a wide range of temperatures and compositions. Although more detailed phase diagrams and precise distinctions between gel and liquid phases are required to fully define the phase behavior of these sterols in vesicles, we find that our classifications of promoter and inhibitor sterols are consistent with previous designations based on fluorescence quenching and detergent resistance. We find no trend in the liquid-liquid or gel-liquid transition temperatures of membranes with promoter or inhibitor sterols and measure the surface fraction of coexisting phases. We find that the vesicle phase behavior is related to the structure of the sterols. Promoter sterols have flat, fused rings, a hydroxyl headgroup, an alkyl tail, and a small molecular area, which are all attributes of "membrane active" sterols.     

Coupling between the helix–coil transition and nematic–isotropic transitions in polypeptide solutions

The lattice model of Flory has been extended in order to consider equilibrium between isotropic and nematic phases containing helix–coil type chains. Nearly complete exclusion of coil sequences from the lyotropic nematic phase produces an enhanced cooperativity in the helix–coil transition. In poor solvents this enhancement begins to occur at concentrations typical of some experiments.     

Effect of sorbitol addition on the physicochemical characteristics of starch–fatty acid systems

Aqueous maize starch dispersions (20%) were heated at 100 °C, in the presence of myristic, palmitic or stearic acid potassium salts as well as of sorbitol added at concentrations up to 60% (dry starch). Flow behaviour measurements at 100 °C indicated that interactions took place between the starch–fatty acid systems and sorbitol resulting in viscosity increase which was more pronounced as the sorbitol content increased. Water solubility measurements showed that a major part of sorbitol was easily extracted by excess water whereas sorption experiments revealed that the moisture uptake rate was proportional to sorbitol content of the starch systems examined. Thermomechanical studies indicated that the starch–fatty acid samples containing sorbitol up to 40% exhibited antiplasticizing behaviour. Scanning electron microscopy studies revealed that at sorbitol concentrations over 30%, free sorbitol crystals were formed on the surface of starch–fatty acid samples, whereas the percentage crystallinity as well as the crystallite size of samples were proportional to sorbitol content.     

Indicators of transitions in biological systems

In the face of global biodiversity declines, predicting the fate of biological systems is a key goal in ecology. One popular approach is the search for early warning signals (EWSs) based on alternative stable states theory. In this review, we cover the theory behind nonlinearity in dynamic systems and techniques to detect the loss of resilience that can indicate state transitions. We describe the research done on generic abundance‐based signals of instability that are derived from the phenomenon of critical slowing down, which represent the genesis of EWSs research. We highlight some of the issues facing the detection of such signals in biological systems – which are inherently complex and show low signal‐to‐noise ratios. We then document research on alternative signals of instability, including measuring shifts in spatial autocorrelation and trait dynamics, and discuss potential future directions for EWSs research based on detailed demographic and phenotypic data. We set EWSs research in the greater field of predictive ecology and weigh up the costs and benefits of simplicity vs. complexity in predictive models, and how the available data should steer the development of future methods. Finally, we identify some key unanswered questions that, if solved, could improve the applicability of these methods.     

Effects of cation binding on the thermal transitions in calmodulin

The thermal transitions in different forms of bovine brain calmodulin (0, 1, 2, 3 and 4 bound Ca2+ ions per molecule) have been studied by means of microcalorimetry, intrinsic tyrosine fluorescence, circular dichroism and infrared spectroscopy. The heating of the apoprotein from 5 to 110 degrees C induces at least three unfolding transitions. The heating of Ca2+-loaded calmodulin causes at least two structural transitions, one of which occurs at relatively low temperatures, from approx. 30 to approx 50 degrees C. The binding of the biologically significant Ca2+, Mg2+, Na+ and K+ ions has been measured at 12, 20, 28, 37 and 50 degrees C by means of the fluorescence method. The values of the binding parameters for these cations do not depend on temperature within the range 12 to 50 degrees C. It has been proposed that the temperature independence of the metal-ion-binding properties of calmodulin is achieved due to the temperature-induced structural changes, which adjust the protein conformation in such a way that the protein-binding parameters remain constant.     

Mechanistic studies of displacer–protein binding in chemically selective displacement systems using NMR and MD simulations

A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair α‐chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1‐(4‐chlorobenzyl)piperidin‐3‐aminesulfate and N′1′‐(4‐methyl‐quinolin‐2‐yl)‐ethane‐1,2‐diamine dinitrate, and one non‐selective displacer, spermidine, were selected as model systems to investigate the mechanism of chemically selective displacement chromatography. Saturation transfer difference (STD) NMR was used to directly evaluate displacer–protein binding. The results indicated that while binding occurred between the two chemically selective displacers and the more hydrophobic protein, α‐chymotrypsin, no binding was observed with ribonuclease A. Further, the non‐selective displacer, spermidine, was not observed to bind to either protein. Importantly, the binding event was observed to occur primarily on the aromatic portion of the selective displacers. Extensive molecular dynamic simulations of protein–displacer–water solution were also carried out. The MD results corroborated the NMR findings demonstrating that the binding of selective displacers occurred primarily on hydrophobic surface patches of α‐chymotrypsin, while no significant long term binding to ribonuclease A was observed. The non‐selective displacer did not show significant binding to either of the proteins. MD simulations also indicated that the charged amine group of the selective displacers in the bound state was primarily oriented towards the solvent, potentially facilitating their interaction with a resin surface. These results directly confirm that selective binding between a protein and displacer is the mechanism by which chemically selective displacement occurs. This opens up many possibilities for future molecular design of selective displacers for a range of applications. Biotechnol. Bioeng. 2009;102: 1428–1437. © 2008 Wiley Periodicals, Inc.     

Disorder–order transitions induced in anionic homopolypeptides by cationic detergents

CD spectra have been obtained for poly(L -glutamic acid) and poly(L -aspartic acid) as functions of temperature and concentration of cationic detergents. Dodecylammonium chloride induces a coil–helix transition in fully ionized poly(L -glutamic acid). The interaction of the monomeric detergent with the polypeptide is responsible for the conformational transition. The detergent concentration required to produce the transition is independent of temperature. The CD of fully ionized poly(L -aspartic acid) is nearly unaffected by dodecylammonium chloride, in marked contrast to the situation found with poly(L -glutamic acid). However, these results do not imply that dodecylammonium chloride interacts differently with aspartyl and glutamyl residues. The observed results can be accounted for by the well-known fact that the glutamyl residue has a higher helix-forming tendency that the aspartyl residue. Cetyltrimethylammonium chloride destabilizes the helical form of poly(L -glutamic acid). This detergent presents an exception to the usual ability of ionic detergents to promote formation of ordered structures in oppositely charged homopolypeptides.     

Multivalent ligand–receptor binding on supported lipid bilayers

Fluid supported lipid bilayers provide an excellent platform for studying multivalent protein–ligand interactions because the two-dimensional fluidity of the membrane allows for lateral rearrangement of ligands in order to optimize binding. Our laboratory has combined supported lipid bilayer-coated microfluidic platforms with total internal reflection fluorescence microscopy (TIRFM) to obtain equilibrium dissociation constant (K_D) data for these systems. This high throughput, on-chip approach provides highly accurate thermodynamic information about multivalent binding events while requiring only very small sample volumes. Herein, we review some of the most salient findings from these studies. In particular, increasing ligand density on the membrane surface can provide a modest enhancement or attenuation of ligand–receptor binding depending upon whether the surface ligands interact strongly with each other. Such effects, however, lead to little more than one order of magnitude change in the apparent K_D values. On the other hand, the lipophilicity and presentation of lipid bilayer-conjugated ligands can have a much greater impact. Indeed, changing the way a particular ligand is conjugated to the membrane can alter the apparent K_D value by at least three orders of magnitude. Such a result speaks strongly to the role of ligand availability for multivalent ligand–receptor binding.     

Sequence–function relationships in folding upon binding

Folding coupled to binding is ubiquitous in biology. Nevertheless, the relationship of sequence to function for protein segments that undergo coupled binding and folding remains to be determined. Specifically, it is not known if the well-established rules that govern protein folding and stability are relevant to ligand-linked folding transitions. Upon small ligand biotinoyl-5′-AMP (bio-5′-AMP) binding the Escherichia coli protein BirA undergoes a disorder-to-order transition that results in formation of a network of packed hydrophobic side chains. Ligand binding is also allosterically coupled to protein association, with bio-5′-AMP binding enhancing the dimerization free energy by −4.0 kcal/mol. Previous studies indicated that single alanine replacements in a three residue hydrophobic cluster that contributes to the larger network disrupt cluster formation, ligand binding, and allosteric activation of protein association. In this work, combined equilibrium and kinetic measurements of BirA variants with alanine substitutions in the entire hydrophobic network reveal large functional perturbations resulting from any single substitution and highly non-additive effects of multiple substitutions. These substitutions also disrupt ligand-linked folding. The combined results suggest that, analogous to protein folding, functional disorder-to-order linked to binding requires optimal packing of the relevant hydrophobic side chains that contribute to the transition. The potential for many combinations of residues to satisfy this requirement implies that, although functionally important, segments of homologous proteins that undergo folding linked to binding can exhibit sequence divergence.