Differences in structure and stability between normal and deuterated proteins (phycocyanin)

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔH_d) and the temperature (t_d) of thermal denaturation of normal and deuterated phycocyanins isolated from two blue-green algae, Plectonema calothricoides and Phormidium luridum. Values of t_d in deuterated proteins are about 5°C lower than those in normal proteins. The magnitudes of ΔH_d in deuterated proteins are 18–36% lower than in normal proteins. The heatcapacity change (ΔC_p) in protein unfolding is essentially the same (2 kcal/mol/K) for deuterated and normal proteins within the experimental error. At close to physiological temperature (27°C), the differences in thermodynamic functions in the native and denatured states are much higher in normal proteins than in deuterated proteins. CD was employed to evaluate both the secondary structures and urea denaturation of these two types of proteins. In P. luridum, deuterated protein is about 8% higher in α-helix content; in P. calothricoides it is not significantly higher. Deuterated proteins are less resistant to the denaturant urea than are normal proteins: the denaturant concentration at the midpoint of the denaturation curve is 0.6–1.2 mol/L lower in the deuterated proteins. The apparent free energies of unfolding of deuterated proteins at zero denaturant concentration are 1.1–1.5 kcal/mol less than for normal proteins.     

Thermal perturbation difference spectra and D2O vs H2O isothermal difference spectra of protein-model aromatic chromophores.

The thermal perturbation difference spectra of phenolic and indolic chromophores in water resemble the isothermal D₂O and H₂O spectra of these chromophores. For phenols approximately equal Δ? values are obtained in both types of spectra, but for their methyl ethers Δ? values of D₂O vs H₂O spectra are about half of those of the thermal perturbation spectra. Phenols and their methyl ethers were studied in deuterated ethylene glycol and glycerol vs the corresponding protiated solvent, and in nonprotic solvents containing 0.25–4% D₂O or H₂O. For phenols in D₂O vs H₂O, about one-third to one-half of the difference spectrum is attributed to solvent structure difference, and the remainder to the effects of replacing OH by OD and to differences in accepting hydrogen bonds from D₂O and H₂O. The refractive index difference between D₂O and H₂O was shown to be a minor contribution by means of experiments in which D₂O was at 5 dgC and H₂O at 47 dgC, conditions of equal refractive index (Na_D). D₂O vs H₂O and glycerol-d vs glycerol-h difference spectra of ribonuclease are about twice as large as expected from the known number of exposed tyrosyl side chains. Possible sources of error in D₂O vs H₂O spectra of proteins are discussed.     

Nucleic acid-solvent interactions: temperature dependence of the heat of solution of thymine in water and ethanol

The heats of solution of thymine in water and ethanol have been determined calorimetrically as a function of temperature. These data, along with solubility data, have been used to calculate the thermodynamic quantities (ΔG_t, ΔH_t, ΔS_t and ΔC_p,t) associated with the transfer of thymine from ethanol to water. Since ΔS_t = ?2 cal/mole deg and ΔC_p,t = 0, it has been concluded that hydrophobic bonding does not play an important role in the thermocynamic stability of nucleic acids. However, large heat capacities of solution of thymine are observed in both solvents (ΔC°_p2 = 45 ± 4 cal/mole deg). This is explained in terms of temperature variation in the degree of solvent–solute hydrogen bonding. It is our proposal that the components of macromolecules (i.e., nucleic acid bases and amino acids) do not make all possible hydrogen bonds with the solvent in the vicinity of room temperature. Thus the thermodynamic contribution of hydrogen bonding to the stability of macromolecules in aqueous solution must be reassessed.     

Stability characteristics of deuterated myoglobin

Purified sperm whale myoglobin was deuterated by being exposed to pD 3.5 in D₂O buffer for 1 hr, then raised to pD 10.6 for an additional hour, and finally brought to neutrality in a D₂O environment. Control myoglobin was similarly treated in H₂O. The specific rotation at 233 mμ and/or the absorbance in the Soret region were used to follow the helix-coil transition of myoglobin subjected to denaturation by acid, alkali, urea and guanidine. Deuterated and control myoglobin had similar 50% transition points in the four denaturing media studied (acid: pH 4.4, pD 4.9; alkali: pH 9.4, pD 10.0; urea, 7.2M; guanidine, 2.5M). The shift toward the alkaline side of 0.5 or 0.6 units of the transition induced by either acid or alkaline denaturation reflects only the weakened acidity of ionizable groups in deuterium systems. Deuterated myoglobin in 3.25M guanidine had a 20% faster denaturation rate than that of control. Acid, urea, and guanidine denaturation curves showed fairly steep transitions, taken as indicative of a one-step process involving only two states (native and denatured molecules). Supporting this conclusion was the fact that plots of absorption and polarimetry measurements of the helix-coil transition induced by either acid or guanidine could be superimposed.     

Evidence for stereospecific collision complexes of cyclo(tri-L-prolyl) with benzene

NMR spectra of cyclo(tri-L -prolyl), c-(P)₃, show large shifts of the H_α resonance on adding C₆D₆ to a solution of c-(P)₃ in CD₂Cl₂. CPK models and observed coupling constants indicate a rigid c-(P)₃ conformation, independent of solvent composition, suggesting that these shifts result from formation of stereospecific C₆D₆–c-(P)₃ collision complexes in which the c-(P)₃ H_α lie near the face of the aromatic ring. The temperature dependence of the H_α shifts and the solvent dependent shifts observed on adding toluene-d₈ or nitrobenzene-d₅ to the c-(P)₃ solution suggest that preferred C₆D₆–c-(P)₃ orientations result from attractive interactions between the electron-rich aromatic ring and the electropositive H_α's and/or δ⁺ nitrogen atoms in the peptide backbone. Reports of such interactions in increasingly diverse peptide model systems suggest that they may play a role in stabilizing protein structures.     

Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts

The endotherm enthalpy changes ΔH_D and temperatures T_D of thermal denaturation of tropocollagen fibers were measured by DSC calorimetry as functions of water content. The denaturation temperatures decrease with increasing water content. The enthalpy change values increase sharply in the range 0–28% of water content, where a maximum of 14.3 cal g^?1 is reached. The effect of water uptake on the enthalpy term is explained by water bridge formation within the collagen triple helix. Evidence is given for the existence of approximately three intercatenary water bridges per triplet at the enthalpy maximum, their H-bond energy amounting to approximately 4000 kcal/mol of protein. In the 30–60% range of water content, ΔH_D decreases by 2 cal^?1 probably due to interactions between secondary water structures and the stabilizing intrahelical water bonds. The influence of two neutral potassium salts, with a structure-stabilizing and a structure-breaking anion (F^? and I^?), on the hydration dependence of ΔH_D and T_D was also studied. It was shown that the primary hydration is not influenced by these ions, but that T_D and ΔH_D are altered in an ion specific way in the presence of interface and bulk water. Hydrophobic interactions do not explain the experimental results. A reaction mechanism of the effects of ions upon the structural stability of collagen is proposed and discussed in terms of interactions of the medium water molecules with the intrahelical water bonds, and in terms of proton-donor/proton-acceptor equilibria between peptide groups, hydrated ions, and intrahelical water molecules.     

Estimation of the stability of globular proteins

The interpretation of ΔG (the free energy change for the reaction, globular conformation ? randomly coiled conformation, in the absence of denaturant), in terms of the free energies of transfer of various parts of the protein molecule from water to denaturant solution, is unsatisfactory because the latter are assumed to be identical to the transfer-free energies of similar groups attached to smaller model compounds. We have made empirical adjustments to transfer-free energy theory that make possible linear extrapolation of the free energy of denaturation of a protein from transition region to zero denaturant concentration. The modified theory, used to analyze the denaturation of proteins by guanidine hydrochloride and urea, allowed us to calculate reasonable values for Δα, the average change in accessibility to solvent of the component groups of protein.     

NMR studies on puerarin and its interaction with beta-cyclodextrin

The interaction between puerarin and β-cyclodextrin (CD) has been studied in D₂O, H₂O/acetone-d ₆, acetone-d ₆ and DMSO-d ₆ solutions by ¹H NMR spectroscopy. The NMR data obtained from hydroxy protons indicate that the formation of the inclusion complex between the two molecules is not stabilized by strong hydrogen bond interactions. The sugar part of puerarin as well as the A ring are outside the β-CD cavity while the B and C rings are located inside the cavity and the interaction is mainly stabilized by hydrophobic interactions. In DMSO at 30°C and in acetone-d ₆/H₂O at temperature below −5°C, doubling of some signals indicated that, in these solvent systems, free rotation of the C-glycosyl bond was restricted due to the steric hindrance between the phenolic hydroxy group at C-7 and the bulky sugar moiety at C-8. In acetone, fast exchange of phenolic protons on the NMR timescale was observed, showing the effect of the solvent on the hindered rotation.     

Glycine betaine may have opposite effects on protein stability at high and low pH values

The compatible osmolyte glycine betaine (GB) is the most efficient osmoprotectant and best excluder from the protein surface. It can reverse protein aggregation and correct mutant protein defects and counter the harmful effects of urea and salts in vivo and in vitro. In this study we have investigated the pH dependence of the stabilizing effect of GB on three different proteins, namely, α-lactalbumin (α-LA), lysozyme and ribonuclease-A (RNase-A). We show here that (a) GB stabilizes RNase-A at all pH values, and (b) GB has opposite effects on two proteins at high pH and low pH values, namely, α-LA and lysozyme. This conclusion was reached by determining T_m (midpoint of denaturation), ΔH_m (denaturational enthalpy change at T_m), ΔC_p (constant-pressure heat capacity change) and ΔG_D^o (denaturational Gibbs energy change at 25 °C) of proteins in the presence of different GB concentrations. Another conclusion of this study is that ΔH_m and ΔC_p are not significantly changed in the presence of GB. This study suggests that other methylated glycine osmolytes may also behave in the same manner.     

Mixing performance of air-lift fermenters against working volume and draft tube dimensions

Mixing characteristics of a laboratory scale internal loop air-lift fermenter has been investigated. The effects of different draft tube dimensions and positions as well as varying levels of liquid height over the draft tube, on mixing time were determined. The results indicate the existance of an optimum liquid height and thus liquid volume with respect to mixing performance especially for the taller draft tubes.List of Symbols A mm distance between draft tube and reactor base - A _D mm² area of the downcomer region - A _R mm² area of the riser region - B mm width of annulus - D _d mm draft tube diameter - D _t mm fermenter diameter - H _d mm draft tube height - H _l mm liquid height in the fermenter - H _t mm fermenter height - V _d m³ draft tube volume - V _t m³ fermenter volume - D _d /D _l - _B H _d /H _l - _F H _l /D _t      

A simple model for determining affinity from irreversible thermal shifts

Thermal denaturation (Tm) data are easy to obtain; it is a technique that is used by both small labs and large‐scale industrial organizations. The link between ligand affinity (K _D) and ΔTm is understood for reversible denaturation; however, there is a gap in our understanding of how to quantitatively interpret ΔTm for the many proteins that irreversibly denature. To better understand the origin, and extent of applicability, of a K _D to ΔTm correlate, we define equations relating K _D and ΔTm for irreversible protein unfolding, which we test with computational models and experimental data. These results suggest a general relationship exists between K _D and ΔTm for irreversible denaturation.     

Raman spectra of polypeptides containing L-histidine residues and tautomerism of imidazole side chain

Raman spectra were measured for poly(L -histidine) in H₂O, poly(L -histidine-d₂ and -d₃) in D₂O, L -histidine in H₂O, L -histidine-d₃ (and d₄) in D₂O, and 4-methylimidazole in H₂O with various pH (or pD) values. The Raman scattering peaks observed for these samples were ascribed to the neutral and positively charged imidazole groups on the basis of the spectral changes due to the pH variation and to the deuterium substitution of the imino protons. The vibrational modes of these peaks were deduced from the normal coordinate analysis made on the positively charged and neutral 4-ethylimidazoles. The Raman scattering peaks from the imidazole groups in the neutral form clearly indicate that these imidazole groups exist in the equilibrium between the two tautomeric forms, the 1-N protonated from (tautomer I) and the 3-N protonated one (tautomer II). For example, the breathing vibration of the 1-N protonated form is observed at 1282 cm^?1 for L -histidine and at 1304 cm^?1 for 4-methylimidazole, while the breathing vibration of the 3-N protonated form is observed at 1260 cm^?1 for L -histidine and 4-methylimidazole. From the temperature dependence of the relative intensities of the tautomer I peak to that of the tautomer II, it was concluded that the tautomer I is energetically more stable than the tautomer II, and the ΔH value is 1.0 ± 0.3 kcal/mol for L -histidine and 0.4 ± 0.1 kcal/mol for 4-methylimidazole. Poly(L -histidine) with the neutral imidazole side chains shows the amide I peak at 1672 cm^?1, indicating that the sample assumes the antiparallel pleated-sheet structure. Poly(L -Ala⁷⁵L -His²⁵) and poly(L -Ala⁵⁰L -His⁵⁰) were found to take the α-helical and β-form conformations, respectively.     

Stability of β-Lactoglobulin A in the Presence of Sugar Osmolytes Estimated from Their Guanidinium Chloride-Induced Transition Curves

The stabilizing role of Trehalose, Sucrose, Sorbitol and Mannitol as sugar osmolytes and polyols on beta-lactoglobulin A (β-lgA) against its chemical denaturation at pH 2.0 and 25 °C has been explored by means of UV–vis spectroscopy. It has been observed that ΔG _D ^o of β-lgA in the presence of 10% (w/v) Trehalose, Sucrose, Sorbitol and Mannitol is increased. We report that the functional dependence of ΔG _D of proteins in the absence and the presence of sugar osmolytes on denaturant concentration is linear. Trehalose is found to induce remarkable stability of β-lgA against chemical denaturation. Furthermore, we assumed sugar osmolytes do not affect the secondary and tertiary structures of the native and GdnHCl-denatured states.     

Studies on the reactivity of malondialdehyde. I. Effects of acid concentration and solvent composition on deuterium exchange reactions

The rates of deuterium exchange reactions of malondialdehyde (MDA) and deuterated malondialdehyde (MDAd) have been studied as a function of acidity and the content of dimethyl sulfoxide (DMSO) in binary mixtures with D₂O . MDA incorporates deuterium from D₂O solutions in a first-order reaction with a rate constant (k_obs) that depends on the acid concentration. From this dependence, a catalytic constant, k_cat, can be derived (k_cat^MDA = 2.25 × 10⁵M^?s^?1). Similar kinetic behavior was found for MDAd in H₂O solutions, and in this case, k_cat^MDA = 1.56 × 10⁵M^?1s^?1. Results from reactions of MDA and MDAd in identical H₂OD₂O mixtures show that primary and secondary isotope effects are small (k_H/k_D = 1.13) and that solvent isotope effects cause most of the differences found between reactions in D₂O and H₂O. Reactions in binary DMSOd₆D₂O mixtures show a six-fold rate increase as the proportion of DMSOd₆ increases from 50% to 90%. These results also illustrate the relatively high reactivity of MDA at pH values well above its pK_a and the importance of medium composition on its reaction rate.     

Purification, properties, and kinetic observations on the isoenzymes of NADP isocitrate dehydrogenase of maize.

A successful method for the purification of NADP isocitrate dehydrogenase from a plant source, Zea mays, is reported. Two mitochondrial isoenzymes were found and purified to homogeneity by a course of acetone fractionation, bulk exchange on DEAE-cellulose, cellulose hydroxylapatite column chromatography, and continuous elution electrophoresis. The mitochondrial isoenzymes are very similar with respect to kinetic properties, response to solvent perturbation, and temperature dependence of the pH/V relationship of isocitrate dehydrogenation. The Michaelis constant for isocitrate is identical for both isoenzymes. The enzymes have a molecular weight of 81,000 as estimated by permeation chromatography and an isoelectric point of 5.5 as extrapolated from gel-electrophoretic mobilities. Detectable differences are confined to differences in electrophoretic mobilities and heat denaturation. In D₂O the rate of the overall reaction from isocitrate to α-ketoglutarate and CO₂ was about 3.6 times slower than the same reaction in H₂O. Both the forward and reverse reactions, in which isocitrate is dehydrogenated or generated from oxalosuccinate, were observed to decrease by this amount in D₂O. The decarboxylation of oxalosuccinate was found to decrease by only about 25% in D₂O relative to the velocity of the reaction in H₂O. Thus the slow step in the overall reaction must be the initial dehydrogenation step rather than the decarboxylation of oxalosuccinate. The pK of the overall reaction did not change in D₂O as compared to H₂O.     

Transition temperature and enthalpy change dependence on stabilizing and destabilizing ions in the helix–coil transition in native tendon collagen

The transition temperatures t_t and enthalpy changes ΔH in the helix–coil transition of solid tendon collagen soaked in a solution containing one of the following stabilizing or destabilizing agents, HCHO, NaF, NaCl, NaI, NaBr, NaOH, NH₂CONH₂, CaCl₂, MgCl₂, were measured as a function of molar concentration by a calorimetric method. The temperature and the enthalpy changes accompanying the transition behaved in a similar manner: when the t_t was depressed by the presence of ions, similar behaviour was observed in ΔH. Both parameters (t_t and ΔH) increased for HCHO, and decreased for NaF and NaCl at concentrations lower than 0.2 M. Above 0.2 M they increased for NaF and NaCl, and decreased in the presence of the other reagents listed above. The average t_t and the ΔH observed in collagen soaked in water were 63.5°C and 12.3 cal/g, respectively. In addition to the parameters mentioned above, the molar effectiveness of the various reagents was obtained for the cases where there was a linear relationship between the t_t and molar concentration of the reagent in the solution. Since both the t_t and the ΔH were observed to vary, the entropy change (ΔS) accompanying the transition was calculated using thermodynamic relations. In order to explain the ΔS observed as a function of ionic concentration, the thermodynamic relationships have been obtained from a partition function under suitable assumptions. Since the partition function is dependent on the number of hydrogen bonds responsible for collagen stability, the result obtained has been compared with the values predicted by the two most quoted models for collagen. The present study is in accordance with the Ramachandran model for collagen structure, which predicts more than one hydrogen bond per three residues.