A new set of peptide-based group heat capacities for use in protein stability calculations.

The partial molar heat capacities of the tripeptides of the sequence glycyl-X-glycine, where X is one of the amino acids leucine, threonine, glutamine, phenylalanine, histidine, cysteine, proline, glutamic acid or arginine, and of the two tetrapeptides tetraglycine and glycyltryptophanylglycylglycine in aqueous solution over the temperature range 10-100 degrees C have been determined using high sensitivity scanning microcalorimetry. These results were used to derive the partial molar heat capacities of the various amino acid side-chains. This report completes our programme to derive reliable side-chain heat capacities for all 20 amino acids of proteins over a wide temperature range using the tripeptides Gly-X-Gly as realistic model compounds. Included in the study is a summary of the partial molar heat capacities of all 20 amino acid side-chains. These results, along with the heat capacity of the peptide backbone group, were used to calculate the partial molar heat capacities of some oligopeptides and of the random coil form of some unfolded proteins in water. The calculated heat capacities of the proteins obtained using this new set of heat capacities for the constituent groups are consistent with the heat capacities of the denatured state determined experimentally.     

Partial molar heat capacities of the peptides glycylglycylglycine, glycyl-L-alanylglycine and glycyl-DL-threonylglycine in aqueous solution over the temperature range 50 to 125 degrees C

Partial molar heat capacities of the three tripeptides glycylglycylglycine, glycyl-L-alanylglycine and glycyl-DL-threonylglycine in aqueous solution at the temperatures 50, 75, 100 and 125 degrees C have been determined using differential flow calorimetry. The results have been used to estimate the contributions to the partial molar heat capacities of peptides of the alanyl and threonyl side-chains. These side-chain contributions are compared with those reported in the literature.     

Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects

Using the heat capacity values for amino acid side-chains and the peptide unit determined in the accompanying paper, we calculated the partial heat capacities of the unfolded state for four proteins (apomyoglobin, apocytochrome c, ribonuclease A, lysozyme) in aqueous solution in the temperature range from 5 to 125 degrees C, with an assumption that the constituent amino acid residues contribute additively to the integral heat capacity of a polypeptide chain. These ideal heat capacity functions of the extended polypeptide chains were compared with the calorimetrically determined heat capacity functions of the heat and acid-denatured proteins. The average deviation of the experimental functions from the calculated ideal ones in the whole studied temperature range does not exceed the experimental error (5%). Therefore, the heat-denatured state of a protein, in solutions with acidic pH preventing aggregation, approximates well the completely unfolded state of this macromolecule. The heat capacity change caused by hydration of amino acid residues upon protein unfolding was also determined and it was shown that this is the major contributor to the observed heat capacity effect of unfolding. Its value is different for different proteins and correlates well with the surface area of non-polar groups exposed upon unfolding. The heat capacity effect due to the configurational freedom gain by the polypeptide chain was found to contribute only a small part of the overall heat capacity change on unfolding.     

Hydration of diglycyl tripeptides with non-polar side chains: a volumetric study

We have determined the apparent molar volumes and the apparent molar adiabatic compressibilities at 25 degrees C of 10 X-Gly-Gly and Gly-Gly-X tripeptides in which X represents a residue with a non-polar side chain. We also have determined the changes in volume and compressibility which accompany neutralization of the amino and carboxyl termini in these tripeptides. The mutual influence of the non-polar side chain of the X residue and the terminal amino and carboxyl groups on the hydration of each other depends on the chemical nature of the side chain and the state of ionization of the termini. We interpret our data in terms of the hydration of the component aliphatic, aromatic, and charged atomic groups, as well as the mutual interactions between these groups.     

Heat capacity analysis of oxidized Escherichia coli thioredoxin fragments (1--73, 74--108) and their noncovalent complex. Evidence for the burial of apolar surface in protein unfolded states.

We have calculated the absolute heat capacities of fragments 1--73 (N fragment) and 74--108 (C fragment) from thioredoxin, their complex and the uncleaved protein, from the concentration dependence of the apparent heat capacities of the solutions determined by differential scanning calorimetry. We find that, while the absolute heat capacities of uncleaved, unfolded thioredoxin and the C fragment are in good agreement with the theoretical values expected for fully solvated chains (calculated as the sum of the contributions of the constituent amino acids), the absolute heat capacities of the N fragment and the unfolded complex are about 2 kJ x K(-1) x mol(-1) lower than the fully solvated-chain values. We attribute this discrepancy to burial of the apolar surface in the N fragment (as burial of the polar area is expected to lead to an increase in heat capacity). Illustrative calculations suggest that burial of about 1000--1600 A(2) of apolar surface takes place in the N fragment (probably accompanied by the burial of a smaller amount of polar surface). In general, this work is supportive of heat capacity measurements on protein fragments being useful as probes of surface burial in studies to characterize protein unfolded states and the high regions of protein folding landscapes.     

Heat capacity and conformation of proteins in the denatured state

Heat capacity, intrinsic viscosity and ellipticity of a number of globular proteins (pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, myoglobin and cytochrome c) and a fibrillar protein (collagen) in various states (native, denatured, with and without disulfide crosslinks or a heme) have been studied experimentally over a broad range of temperatures. It is shown that the partial heat capacity of denatured protein significantly exceeds the heat capacity of native protein, especially in the case of globular proteins, and is close to the value calculated for an extended polypeptide chain from the known heat capacities of individual amino acid residues. The significant residual structure that appears at room temperature in the denatured states of some globular proteins (e.g. myoglobin and lysozyme) at neutral pH results in a slight decrease of the heat capacity, probably due to partial screening of the protein non-polar groups from water. The heat capacity of the unfolded state increases asymptotically, approaching a constant value at about 100 degrees C. The temperature dependence of the heat capacity of the native state, which can be determined over a much shorter range of temperature than that of the denatured state and, correspondingly, is less certain, appears to be linear up to 80 degrees C. Therefore, the denaturational heat capacity increment seems to be temperature-dependent and is likely to decrease to zero at about 140 degrees C.     

Living organisms and the quantum-mechanical wave properties of matter

Heat capacity data for the solid chemical elements and for 10 amino acids from proteins, and Kopp's Law heat capacity values for the chemical elements, show that proteins have extremely low heat capacities compared to other substances. The type of quantum-mechanical interaction that is highly predominant in low-heat-capacity substances reveals the quantum wave properties of matter. Therefore, the quantum-mechanical wave properties of matter are highly predominant in proteins. Some speculations about the meaning of this result are examined and assessed.     

Purification of gamma-glutamyltransferase by phenyl boronate affinity chromatography. Studies on the acceptor specificity of transpeptidation by rat kidney gamma-glutamyltransferase

gamma-Glutamyltransferase has been purified from rat kidney by a novel procedure using phenyl boronate affinity chromatography. The highly purified enzyme has been studied with respect to acceptor specificity for a number of amino acids, amino acid analogues, dipeptides and tripeptides. The acceptor activity is specific for L-amino acids. The amino acids and the majority of the essential amino acids are poor acceptors while the sulphur-containing amino acids are the best acceptors. The acceptor activity is modulated by the substitution of the amino acid side chain. Substitution of the side chain at the delta, gamma or beta positions results in a proportionally decreasing ability to act as acceptor. The carbonyl moiety of the gamma-carboxy group of the acceptor appears to be essential for acceptor activity, absence of an alpha-carboxy carbonyl group increases the Kappm of the acceptor approximately 100-fold.     

Heat capacities of transfer of some amino acids and peptides from water to aqueous urea solution

Integral enthalpies of solution at low concentrations of several amino acids and peptides in 2 and 6M urea solutions have been determined at 25 and 35°C. These data have been used to derive the enthalpies of transfer (at 25 and 35°C) and heat capacities of transfer (at 30°C) of these amino acids and peptides from water to aqueous urea solutions. Furthermore, the enthalpies of transfer and heat capacities of transfer per CH₂ group and per peptide group ? CONH? have also been estimated. These results show that while the enthalpies and heat capacities of transfer per CH₂ group are positive and negative, respectively, the reverse is true for ? CONH? group. The implications of these results in the mechanism of the denaturation of proteins by urea are discussed.     

Heat capacity changes and partial molal heat capacities of some di- and tripeptides in water

Integral enthalpies of solution of several dipeptides and tripeptides in water at low concentrations have been determined at 25 and 35°C. These data have been used to derive the changes in heat capacity on dissolution at infinite dilution ΔC at 30°C. Limiting partial molal heat capacities ΔC have been determined by combining ΔC with C_p2 (heat capacity of pure solid peptides). Using the data on ω-amino acids and these peptides, the partial molal heat capacity of a peptide group ? CONH? was semiquantitatively estimated.     

The partial molar heat capacity and volume of the peptide backbone group of proteins in aqueous solution

The partial molar heat capacities have been determined for the series of peptides alanyl(glycyl)(x)glycine, x=1-3, and for the compounds N-acetylglycinamide and N-acetyl glycylglycinamide in aqueous solution over the temperature range 10-100 degrees C using high sensitivity scanning microcalorimetry. The partial molar volumes for these compounds have also been determined over the temperature range from 10 to 90 degrees C using a scanning densimetric method. The results were used to derive the partial molar heat capacities and volumes of the glycyl group at temperatures in the range 10-100 degrees C. The results obtained are critically compared with literature results derived using heat capacity and volume data for some oligoglycines.     

Heat capacities of protein functional groups

Using a precise technique of scanning calorimetry the heat capacities of a series of carboxylic acids and their sodium salts, alcohols, and N-substituted amides have been measured from 5 to 100 degrees C. From these data, the partial molar heat capacities of CH2, CONH, COOH, and COONa groups have been determined. It is shown that the heat capacity of the CH(2) group in aqueous solution is independent of the type of compound used for its determination, is positive at low temperature, and is linearly decreasing in magnitude with an increase in temperature. In contrast, the heat capacities of COOH and COONa groups in aqueous solution are negative at room temperature and their magnitude non-linearly decreases with an increase in temperature. It appears that the partial heat capacity of CONH group in aqueous solution depends on the type of model compound used for its determination. These differences correlate with the difference in the water accessible surface area of atoms in the CONH group in different model compounds.     

Heat capacities of supercritical fluids via Grand Canonical ensemble simulations

In this work, suitable mathematical relationships to compute isobaric heat capacities from molecular simulations in the Grand Canonical (GC) ensemble are derived and tested via Monte Carlo methods. Using atomistic classical force fields, the residual isobaric heat capacities of pure carbon dioxide (CO₂) and pure methanol (MeOH) were obtained at supercritical conditions (with critical properties estimated from a finite-size scaling analysis). The total isobaric heat capacity was determined by combining the residual isobaric heat capacity obtained from molecular simulations with the ideal gas contributions obtained from experimental correlations. Isobaric heat capacities generated from both GC and Isothermal–Isobaric ensemble simulations were compared to predictions from accurate equations of state (EOS)s for CO₂ and MeOH at corresponding reduced temperatures and pressures. Isobaric heat capacities calculated from both ensembles were in good agreement with those obtained from the Span and Wagner EOS for CO₂ and the IUPAC EOS for MeOH. For comparable computation times, simulations run in the GC ensemble generate results with significantly lower statistical uncertainty than those run in the Isothermal–Isobaric ensemble.     

Fmoc-2-mercaptobenzothiazole, for the introduction of the Fmoc moiety free of side-reactions

A double side-reaction, consisting in the formation of Fmoc-beta-Ala-OH and Fmoc-beta-Ala-AA-OH, during the preparation of Fmoc protected amino acids (Fmoc-AA-OH) with Fmoc-OSu is discussed. Furthermore, the new Fmoc-2-MBT reagent is proposed for avoiding these side-reactions as well as the formation of the Fmoc-dipeptides (Fmoc-AA-AA-OH) and even tripeptides, which is another important side-reaction when chloroformates such as Fmoc-Cl is used for the protection of the alpha-amino function of the amino acids.     

Relating structure to thermodynamics: the crystal structures and binding affinity of eight OppA-peptide complexes.

The oligopeptide-binding protein OppA provides a useful model system for studying the physical chemistry underlying noncovalent interactions since it binds a variety of readily synthesized ligands. We have studied the binding of eight closely related tripeptides of the type Lysine-X-Lysine, where X is an abnormal amino acid, by isothermal titration calorimetry (ITC) and X-ray crystallography. The tripeptides fall into three series of ligands, which have been designed to examine the effects of small changes to the central side chain. Three ligands have a primary amine as the second side chain, two have a straight alkane chain, and three have ring systems. The results have revealed a definite preference for the binding of hydrophobic residues over the positively charged side chains, the latter binding only weakly due to unfavorable enthalpic effects. Within the series of positively charged groups, a point of lowest affinity has been identified and this is proposed to arise from unfavorable electrostatic interactions in the pocket, including the disruption of a key salt bridge. Marked entropy-enthalpy compensation is found across the series, and some of the difficulties in designing tightly binding ligands have been highlighted.     

Nucleotide-induced changes in the heat capacity of beef cardiac myosin

The heat of reaction of ATP with beef cardiac myosin has been determined in a flow microcalorimeter at two different temperatures. The reaction heat obtained by extrapolation to infinite flow rate is interpreted to be the heat of formation of the steady-state set of myosin-nucleotide complexes. The large temperature dependence of this heat, an apparent change in heat capacity, could be caused by an isomerization between two myosin conformations. The enthalpies and heat capacities of binding of ADP, AMP, and pyrophosphate have also been measured and are discussed in terms of this model.     

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.     

Partial molar volumes of polypeptides and their constituent groups in aqueous solution over a broad temperature range

The partial molar volumes of various compounds that model protein constituent groups, such as tripeptides (Gly-X-Gly, where X = Gly, Ala, Val, Leu, Ile, Pro, Met, His, Ser), homopeptides (Glyn, n = 3,4,5), and simple organic analogues of amino acid side chains (methanol, acetamide, propanamide, acetic acid, propanoic acid, n-butanamine, n-butanamine nitrate, n-propylguanidine nitrate, 4-methylphenol), have been determined in aqueous solution with a vibrational densimeter in the temperature range of 5-85 degrees C. The partial molar volumes of amino acid side chains and the peptide unit were estimated from the data obtained. Assuming additivity of component groups, the partial molar volumes of polypeptide chains of several proteins over a broad temperature range were calculated. The partial molar volume functions of four proteins (myoglobin, cytochrome C, ribonuclease A, lysozyme) were compared with those determined experimentally for the unfolded and native forms of these proteins. It has been shown that the average deviation of the calculated functions from the experimental ones does not exceed 3% over the temperature range studied.     

Hydration heat capacity of nucleic acid constituents determined from the random network model

The heat capacities of hydration (dCp) of the five nucleic acid bases A, G, C, T, and U, the sugars ribose and deoxyribose, and the phosphate backbone were determined using Monte Carlo simulations and the random network model. Solute-induced changes in the mean length and root mean square angle of hydrogen bonds between hydration shell waters were used to compute dCp for these solutes. For all solutes the dCp is significantly more positive than predicted from accessible surface area (ASA) models of heat capacity. In ASA models, nitrogen, oxygen, and phosphorus atoms are considered as uniformly polar, therefore making a negative contribution to dCp. However, the simulations show that many of these polar atoms are hydrated by water whose hydrogen bonds are less distorted than in bulk, leading to a positive dCp. This is in contrast to the effect of polar groups seen previously in small molecules and amino acids, which increase the water H-bond distortion, giving negative dCp contributions. Our results imply that dCp accompanying DNA dehydration in DNA-ligand and DNA-protein binding reactions may be significantly more negative than previously believed and that dehydration is a significant contributor to the large decrease in heat capacity seen in experiments.     

Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

The conformational cis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that the cis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing the cis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on the cis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L- or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of the cis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bond undergoing the cis/trans isomerisation moves the equilibrium to a significant amount of the cis form.