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.     

Helix–coil transition for poly(L-glutamic acid) in water–ethanol solutions

Potentiometric titrations and some complementary optical rotation data are presented for solutions of poly(L - glutamic acid) (PGA) in several H₂O–ethanol mixtures. The data allow the determination of the intrinsic pK (pK₀), slope of the apparent. pK (pK_app), versus degree of ionization curves and of the enthalpy of ionization as a function of ethanol concentration. The variation of the degree of ionization at which the helix–coil transformation occurs with ethanol and temperature is also determined. Finally free energy, enthalpy, and intropy changes associated with the helix–coil transformation for the uncharged conformers are determined from the titration curves. The effect of the ethanol is to increase the stability of the helical conformation of PGA for both the charged and the uncharged forms of the polymer. The stabilization of the uncharged helix is essentially an entropic effect.     

Helix–coil transition and conformational studies of protamine–DNA complexes

Protamine–DNA complexes prepared by the method of direct and slow mixing in 2.5 × 10^?4M EDTA, pH 8.0, have been studied by thermal denaturation and circular dichroism. The complexes show biphasic melting with T_m at about 50 °C corresponding to the melting of free DNA regions and T_m′ at about 92 °C corresponding to the melting of protamine-bound regions. In protamine-bound regions there are 1.38 amino acid residues per nucleotide, indicating a nearly completely charge neutralization. T_m is increased but T_m′ is not when the ionic strength of the buffer is raised. This also supports a full charge neutralization in protamine-bound regions. The circular dichroism of the complexes can be decomposed into two components, Δ_ε0 of free DNA regions in B-form conformation and Δ_εb of protamine-bound regions in a characteristic conformation neither that of B- nor C-form but somewhere between them.     

Helix–coil transition in nucleoprotein—theory and applications

A general theory of helix–coil transition of irreversibly complexed nucleoproteins is presented. The equations are tested by experimental results in basic polypeptide–DNA complexes, nucleohistone I and pea bud nucleohistones. They show good agreement between theory and experiments. The theory provides direct measurement of a fraction of DNA base pairs covered by proteins, yielding a value of about 75% histone-covered base pairs in pea bud nucleohistone. It also provides a measurement of an average number of amino acid residues per nucleotide in protein-bound regions. This number varies from 1.0 to 1.4 in DNA–polylysine or DNA–polyarginine and from 2.9 to 3.3 in nucleohistone Ia, Ib, f1, and pea bud nucleohistone.     

The behaviour of polyamino acids reveals an inverse side chain effect in amyloid structure formation

Amyloid fibrils and prions are proteinaceous aggregates that are based on a unique form of polypeptide configuration, termed cross-beta structure. Using a group of chemically distinct polyamino acids, we show here that the existence of such a structure does not require the presence of specific side chain interactions or sequence patterns. These observations firmly establish that amyloid formation and protein folding represent two fundamentally different ways of organizing polypeptides into ordered conformations. Protein folding depends critically on the presence of distinctive side chain sequences and produces a unique globular fold. By contrast, the properties of different polyamino acids suggest that amyloid formation arises primarily from main chain interactions that are, in some environments, overruled by specific side chain contacts. This side chain effect can be thought of as the inverse of the one that characterizes protein folding. Conditions including Alzheimer's and Creutzfeldt-Jakob diseases represent, on this basis, pathological cases in which a natural polypeptide chain has aberrantly adopted the conformation that is primarily defined by main chain interactions and not the structure that is determined by specific side chain contacts that depend on the polypeptide sequence.     

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.     

Helix–coil stability constants for the naturally occurring amino acids in water. XXI. Glutamine parameters from random poly(hydroxypropylglutamine-co-L-glutamine) and poly(hydroxybutylglutamine-co-L-glutamine)

Water-soluble, random copolymers containing L -glutamine and either N⁵-(3-hydroxypropyl)-L -glutamine or N⁵-(4-hydroxybutyl)-L -glutamine were synthesized, fractionated, and characterized. The thermally induced helix–coil transitions of these copolymers were studied in water. A short-range interaction theory was used to deduce the Zimm-Bragg parameters σ and s for the helix–coil transition in poly(L -glutamine) in water from an analysis of the melting curves of the copolymers in the manner described in earlier papers. The computed values of s indicate that L -glutamine is helix-indifferent at low temperature and a helix-destabilizing residue at high temperature in water. At all temperatures in the range of 0–70°C, the glutamine residue promotes helix–coil boundaries since the computed value of σ is large.     

Helix–coil transition in heterogeneous chains. II. DNA model

Melting curves are calculated for infinitely long DNA-like random copolymers composed of AT and GC pairs of nucleotides. The entropy of random coil rings formed on melting is explicitly included through use of the Jacobson-Stockmayer ring-weighting factors. Transition curves are calculated for values of the cooperativity parameter σ in the range 10^?2 ? σ ? 10^?4. Ninety percent of the melting occurs in ca. 0.2°C for σ ? 10^?3 regardless of the mole fraction of GC. We conclude that observed breadths of thermal denaturation curves for native DNAs result from a superposition of essentially all-or-none melting of various regions of the molecule. It is argued that refined approximations to the ring-weighting factors are probably not important when compared with the effects produced by long-range base sequence correlations which are known to occur in native DNA.     

Stability of helical polyamino acids in solution at high temperatures

Optical rotatory dispersion studies have been carried out at temperatures up to 150 °C. on poly(γ-benzyl L -glutamate) in α-chloronaphthalene and N-methylcaprolactam, and on poly-ε-carbobenzoxy-L -lysine, poly-δ-carbobenzoxy-L -ornithine, and poly(L -glutamic acid) in N-methylacetamide. The Moffitt-Yang ?b₀ values were large in all cases, but significant decreases in ?b₀ were observed at the upper temperature limits of the study suggesting that a transition region was being entered. Polymer degradation generally precluded examination of the systems through the suggested transition region.     

Light emission from the Fe2+–EDTA–ascorbic acid–H2O2 system strongly enhanced by plant phenolic acids

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra‐weak photon emission (UPE). We investigated the UPE from the Fe²⁺–EDTA (ethylenediaminetetraacetic acid)–AA (ascorbic acid)–H₂O₂ (hydrogen peroxide) system with a multitube luminometer (Peltier‐cooled photon counter, spectral range 380–630 nm). The UPE, of 92.6 μmol/L Fe²⁺, 185.2 μmol/L EDTA, 472 μmol/L AA, 2.6 mmol/L H₂O₂, reached 1217 ± 118 relative light units during 2 min measurement and was about two times higher (P < 0.001) than the UPE of incomplete systems (Fe²⁺–AA–H₂O₂, Fe²⁺–EDTA–H₂O₂, AA–H₂O₂) and medium alone. Substitution of Fe²⁺ with Cr²⁺, Co²⁺, Mn²⁺ or Cu²⁺ as well as of EDTA with EGTA (ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid) or citrate powerfully inhibited UPE. Experiments with scavengers of reactive oxygen species (dimethyl sulfoxide, mannitol, sodium azide, superoxide dismutase) revealed the dependence of UPE only on hydroxyl radicals. Dimethyl sulfoxide at the concentration of 0.74 mmol/L inhibited UPE by 79 ± 4%. Plant phenolics (ferulic, chlorogenic and caffec acids) at the concentration of 870 μmol/L strongly enhanced UPE by 5‐, 13.9‐ and 46.8‐times (P < 0.001), respectively. It is suggested that augmentation of UPE from Fe²⁺–EDTA–AA–H₂O₂ system can be applied for detection of these phytochemicals.     

Metabolomics of urinary organic acids in respiratory chain deficiencies in children

Metabolomic analysis of the urinary organic acids from 39 selected children with defined respiratory chain deficiencies (RCDs) was performed using untargeted gas chromatography–mass spectrometry, revealing the presence of 255 endogenous and 46 exogenous substances. Variable reduction identified 92 variables from the endogenous substances, which could be analysed by univariate and multivariate statistical methods. Using these methods, no characteristic organic acid biomarker profile could be defined of practical value for diagnostic purposes for complex I (CI), complex III (CIII) and multiple complex (CM) deficiencies. The statistical procedures used did, however, disclose 24 metabolites that were practical highly (d > 0.75) and statistically (P < 0.05) significant for the combined and clinically closely related group of RCDs. Several of these metabolites occur in single enzyme inherited metabolic diseases, but most were not previously reported to be linked to the metabolic perturbations that are due to RCDs. Ultimately, we constructed a global metabolic profile of carbohydrate, amino acid and fatty acid catabolism, illuminating the diverse and complex biochemical consequences of these disorders. This metabolomics investigation disclosed a metabolite profile that has the potential to define an extended and characteristic biosignature for RCDs and the development of a non-invasive screening procedure for these disorders.     

Helix–coil stability constants for amino acids in nonaqueous solvents. Studies of random poly(γ-benzyl-L-glutamate-co-L-alanine) and poly(γ-benzyl-L-glutamate-co-L-leucine) in a mixed solvent

The host–guest technique has been applied to the determination of the helix–coil stability constants of two naturally occurring amino acids, L -alanine and L -leucine, in a nonaqueous solvent system. Random copolymers containing L -alanine and L -leucine, respectively, as guest residues and γ-benzyl-L -glutamate as the host residue were synthesized. The polymers were fractionated and characterized for their amino acid content, molecular weight, and helix–coil transition behavior in a dichloroacetic acid (DCA)–1,2-dichloroethane (DCE) mixture. Two types of helix–coil transitions were carried out on the copolymers: solvent-induced transitions in DCA–DCE mixtures at 25°C and thermally induced transitions in a 82:18 (wt %) DCA–DCE mixture. The thermally induced transitions were analyzed by statistical mechanical methods to determine the Zimm-Bragg parameters, σ and s, of the guest residues. The experimental data indicate that, in the nonaqueous solvent, the L -alanine residue stabilizes the α-helical conformation more than the L -leucine residue does. This is in contrast to their behavior in aqueous solution, where the reverse is true. The implications of this finding for the analysis of helical structures in globular proteins are discussed.     

Dissection of nucleophilic and acid–base catalysis in glycosidases

A startling array of added anions have been observed to function as replacement catalytic nucleophiles in mutant glycosidases, including formate, azide, fluoride and other halides. Likewise, the mechanism of acid–base catalysis is somewhat plastic. The carboxylic acids can be substituted by a sulfenic acid or by ascorbate, and the effective acid strength enhanced by the introduction of strong hydrogen bonds. These studies provide an interesting bridge between enzymes and models thereof.     

Changes in the Mark–Houwink hydrodynamic volume of chitosan molecules in solutions of different organic acids, at different temperatures and ionic strengths

The objective of this study is to explore the cause(s) of changes in the hydrodynamic volume of chitosan molecules in solutions of different organic acids, at different temperatures and ionic strengths. Change in intrinsic viscosity is used as the parameter to elucidate the causes of changes in the hydrodynamic volume of chitosan molecules in these solutions. Results show that the intrinsic viscosity of chitosan decreases in acetic acid or in malic acid over storage time. These decreases are more pronounced in acetic acid solution than in malic acid solution, more significant in higher temperature than in lower temperature solutions, and greater in solutions without NaCl than in solutions containing higher NaCl. The decrease in intrinsic viscosity can perhaps be attributed to the compounded effects of compaction of the chitosan molecules and/or acidic degradation during storage.     

The role of organic acids in assisted phytoremediation processes of salt marsh sediments

Some plants have high ability to absorb heavy metals in high concentrations. In this study, Spartina maritima was tested in conjunction with low molecular weight organic acids (LMWOA), in order to evaluate the possible use of this plant in phytoremediation processes in salt marshes. Three different LMWOA (citric acid, malic acid and acetic acid) were applied to contaminated intact cores of S. maritima colonized sediment and several heavy metals (Cd, Zn, Pb, Cu, Cr and Ni) were analyzed in sediment and plant parts. Acetic acid application proved to be the most efficient, enhancing greatly the uptake of all metals analyzed. Citric acid also showed good results, while malic acid proved to be very inefficient in most of the cases. The highest enhancement was observed for Cr with a 10-fold increase of the uptake upon application of acetic acid, while improving the Pb uptake proved to be the most difficult, probably due to its low solubility.