High-sensitivity Scanning Microcalorimetry Research Articles

Additivity of the partial molar heat capacities of the amino acid side-chains of small peptides: Implications for unfolded proteins

The partial molar heat capacities at infinite dilution, Cp,20, for eight tetrapeptides of sequence glycyl–X–Y–glycine and four pentapeptides of sequence glycyl–X–Y–Z–glycine, where X, Y and Z are amino acids with neutral side-chains, have been determined in aqueous solution over the temperature range 283.15 to 373.15 K using high sensitivity scanning microcalorimetry. The results are compared with those calculated by group additivity using the partial molar heat capacities for the constituent groups of unfolded proteins that we reported in previous work. The comparison verifies that the heat capacity of a polypeptide with neutral side-chains can be reliably estimated using the principle of group additivity and our published group heat capacities.

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 °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.

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) xglycine, x=1–3, and for the compounds N-acetylglycinamide and N-acetyl glycylglycinamide in aqueous solution over the temperature range 10–100°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°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°C. The results obtained are critically compared with literature results derived using heat capacity and volume data for some oligoglycines.

Tripeptides in aqueous solution: Model compounds for the evaluation of the partial molar heat capacities of amino acid side-chains in proteins

The partial molar heat capacities of the tripeptides of sequence glycyl-X-glycine, where X is one of the amino acids glycine, alanine, valine, serine, tyrosine, lysine and aspartic acid, in aqueous solution have been determined over the 10–100°C range, using high sensitivity scanning microcalorimetry. Using these results, the partial molar heat capacities of the various amino acid side-chains were derived. The values obtained differ significantly from literature data that were derived using partial molar heat capacities for small organic compounds that model the various amino acid side-chains. A rationale has been proposed to account for the observed differences between the side-chain heat capacities derived using partial molar heat capacity data for the tripeptides and those derived using the partial molar heat capacities of the organic analogues.

Conformational stability of adrenodoxin mutant proteins.

Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.

Differential scanning calorimetric study of the thermal stability of xylanase from Streptomyces halstedii JM8.

The thermal stability of two xylanases with molecular masses of 45 (Xys1L) and 35 (Xys1S) kDa has been characterized thermodynamically by high-sensitivity scanning microcalorimetry in the pH range 3.0-9.0. Thermal denaturation of Xys1L reveals three thermodynamically independent domains, and that of Xys1S, which is a proteolytic fragment of Xys1L (without a C-terminal part), reveals two thermodynamically independent domains, each of which follows a two-state thermal unfolding process under our experimental conditions. Nevertheless, the thermodynamic parameters of unfolding for each domain do not fit some of the correlations obtained for most compact globular proteins. It is known that if delta Hres(T) and delta Sres(T) are plotted against temperature for a number of water-soluble compact globular proteins, they all have a common value at approximately 110 degrees C (383 K). Calculation of the variations in the enthalpy and entropy of unfolding per residue for each domain of xylanase with temperature gave us delta Hres(383) and delta Sres(383) values of approximately 3 kcal/(mol of residue) and 9 cal/(K.mol of residue), respectively. This is practically 2-fold larger than those apparent for most medium-sized globular protein values. These discrepancies might be related to features of the folded and/or unfolded states of the protein.

Thermodynamics of unfolding of the α-amylase inhibitor tendamistat: Correlations between accessible surface area and heat capacity

Unfolding of the small α-amylase inhibitor tendamistat (74 residues, 2 disulfide bridges) has been characterized thermodynamically by high sensitivity scanning microcalorimetry. To link the stability parameters with structural information we use heat capacity group parameters and water accessible surface areas to calculate the change in heat capacity on unfolding of tendamistat. Our results show that both the group parameter and surface area approaches provide a reasonable, though not perfect, basis for ΔC p calculations. When using the experimentally determined temperature-independent heat capacity increase of 2.89 kJ mol −1 K −1 tendamistat exhibits convergence of thermodynamic parameters at about 140 °C, in agreement with recent predictions of the temperature at which the hydrophobic hydration is supposed to disappear. Despite the apparent support of this new view of the hydrophobic effect, there are inconsistencies in the interpretation of the thermodynamic parameters and these are addressed in the Discussion. The specific stability of tendamistat is similar to that of modified bovine pancreatic trypsin inhibitor, with only two of the native three disulfide bridges intact. This observation confirms our previous conclusion that disulfide bridges affect significantly the enthalpy and entropy of unfolding. The recent study by Doig & Williams provides additional convincing support for this conclusion. The predictive scheme proposed by these authors permits a fair estimate of the Gibbs free energy and enthalpy changes of these two proteins.

Punktmutationen und Proteinstabilität

Proteins are characterized by a marginal stability of their secondary and tertiary structure that renders them susceptible to stability perturbations by single amino acid exchanges. One of the most powerful techniques to determine the typical thermodynamic stability parameters is high sensitivity scanning microcalorimetry. The present paper describes some basic methodological aspects of the technique. Main attention is, however, focussed on the determination and magnitude of covalent and noncovalent contributions to overall protein stability. Two well defined proteins, bovine pancreatic trypsin inhibitor /BPTI/ and /ROP/ have served to exemplify the general rationale of the approach.

The effect of the detergent cetyltrimethylammoniumchloride on the phase behaviour of dipalmitoylphosphatidylcholine: A high precision calorimetric study.

The influence of the linear, positively charged detergent cetyltrimethylammoniumchloride (CTAC1) on the well known phase behaviour of fully hydrated dipalmitoylphosphatidylcholine (DPPC) has been studied along fourteen isopleths of the phase diagram using high sensitivity scanning microcalorimetry. Particular interest was focused towards the effects of very small amounts of detergent down to the region of 1 CTAC1 per 10 4 DPPC-molecules. Even starting with such low concentrations distinct changes of the thermograms are detectable by high sensitivity DSC. Essentially, the phase diagram is cut into four domains between the two limiting cases of pure phospholipid multilamellar dispersion and isotropic solution of detergent micelles, respectively. At the present stage these four regions in the phase diagram are identified as follows : 1. 0–1 Mol % CTAC1: a modified lamellar phase 2. 1–50 Mol % CTAC1: a two-phase region with this modified lamellar phase coexisting with a newly formed detergent-rich phase. 3. 50–90 Mol % CTAC1: large mixed micelles. 4. > 90 Mol % CTAC1: an isotropic solution of small mixed phospholipid/detergent micelles.