Steady-state flow-mix calorimetric methods require large amounts of sample and time to obtain data at variable reactant ratios. The use of continuous concentration gradients allows rapid generation of data with chemicals consumption comparable to other calorimetric techniques. An experimental set-up able to generate a linear concentration gradient of one reactant, while keeping the other concentration constant during the experiment, permits a complete titration in a single experiment. Since the calorimeter is not operating under steady-state conditions, the output is affected by the dynamics of the instrument. Therefore, it is necessary to correct the experimental curve by deconvolution to obtain the true signal. Titration of the carboxyl group of glycine with hydrochloric acid, as well as the binding of ribonuclease A to two different nucleotides prove the efficacy of this new methodology.

For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of α-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above ∼0.5mM the population of the intermediate state becomes significant at temperatures between 45°C and 80°C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.

We have studied the effects produced by site‐directed mutagenesis upon energetic and structural cooperativity in the Src homology region 3 domain of α‐spectrin. The mutation of Asn47 to Gly or Ala in the distal loop brings about significant changes to the global stability of the domain in spite of not affecting its structure to any great extent. The binding affinity for a proline‐rich peptide is also largely diminished in both mutant domains. We have compared the apparent Gibbs energies of the amide hydrogen–deuterium exchange (HX) between the wild‐type and the Gly47 mutant. The observed changes in the Gibbs energy of HX indicate a remarkable energetic cooperativity in this small domain. Regions of the domain's core have a high cooperativity with the position of the mutation, indicating that their HX occurs mainly in states in which the distal loop is unstructured. More flexible regions, which undergo HX mainly by local motions, show a lower but still considerable cooperativity with the distal loop. We conclude that there is an important correlation between regional stability and cooperativity in this small domain.

Using nuclear magnetic resonance we have measured the hydrogen exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin as a function of pH*. At very acidic pH* values the exchange of most residues appears to occur via global unfolding, although several residues show abnormally large Gibbs energies of exchange, suggesting the presence of some residual structure in the unfolded state. At higher pH* HX occurs mainly via local or partial unfoldings. We have been able to characterize the coupling between the electrostatic interactions in this domain and the conformational fluctuations occurring under native conditions by analyzing the dependence upon pH* of the Gibbs energy of exchange. The SH3 domain seems to be composed of a central core, which requires large structural disruptions to become exposed to the solvent, surrounded by smaller subdomains, which fluctuate independently.

The amide hydrogen-deuterium exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin has been measured by nuclear magnetic resonance as a function of temperature between 8 and 46 degrees C. The analysis of the temperature dependence of HX from a statistical thermodynamic point of view has allowed us to estimate the enthalpies and entropies of the conformational processes leading to HX. The results indicate that under native conditions the domain undergoes a wide variety of conformational fluctuations, ranging from local motions, mainly located in loops, turns and chain ends and involving only low enthalpy and entropy, to extensive structural disruptions affecting its core and involving enthalpies and entropies that come fairly close to those observed during global unfolding.

In a previous article a comprehensive description of an isothermal titration microcalorimeter with Peltier compensation was reported. This work deals with the characterization procedure and the operation mode. The transfer function parameters (time constants, calibration constants, and thermal properties of the system components) have been determined using a rigorous physical model for the microcalorimeter. To check the good performance of the instrument, titration experiments of cytidine and adenosine protonation have been carried out. Finally, as an example of the instrument applicability, differential heat measurements of myoglobin adsorption onto polymeric (polystyrene) latex particles are presented.

A detailed description of a differential isothermal titration microcalorimeter with Peltier compensation is reported here. It can be considered as a heat flux or conduction microcalorimeter with dynamic power compensation. The design, based on the twin principle, is very simple and the manufacture is of low cost and complexity. The system operation is completely automatized and computer controlled, including the data acquisition tasks, the controller of the power compensation feedback loop and the reagent injection system. The purpose of the closed loop control is to improve the dynamic performance of the microcalorimeter, resulting in an enhanced sensitivity and accuracy. The instrument performance is demonstrated by calibration experiments showing that the baseline noise (root-mean-square of the temperature difference between the cells) to be 0.4 μK and the specific sensitivity about 2–8 μJ/ml. The main time constant can be reduced from 120 s (without power compensation) to 22 s or even less (with power compensation). In a subsequent article, a precise characterization of the microcalorimeter will be given.

An old problem in Isothermal Titration Calorimetry is the accurate characterisation of the instrument, i.e. the determination of the instrumental transfer function. Normally, this calibration is performed electrically or through well known chemical reactions, but the transfer function parameters obtained by both methods generally do not agree: the first method normally yields smaller time constants than the second one. This fact is explained by the different path the heat flux takes towards the thermal sink. However, the time constants must be independent of the experiment type (electrical or chemical). In order to attain this independency, a realistic physical model of the system is developed taking into account the different heat sources and the paths in the system and using physically attainable (experimental and theoretical) inputs for testing the model. Important results from the model study are that the instrument is represented by different transfer functions, depending on the heat source location, and that the time constants are the same, regardless of the heat source location. A very simple and fast method based on such non-phenomenological physical model for obtaining the transfer function of an Isothermal Titration Microcalorimeter is applied here.

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.

This paper introduces a new measuring method in Isothermal Titration Calorimetry (ITC). This method, based on a proportional-integral control system, offers many important advantages when compared to the frequently used proportional control: higher signal-to-noise ratio, improved stability of baseline, increased static and dynamic sensitivity, similar or smaller time constants and calibration constant independent of the control parameters. Here an experimental proof of this method is detailed and full theoretical foundation will be discussed in a next paper.

The energetics of the interaction between liver glycogen phosphorylase b and the adenosine 5'-monophosphate (AMP) have been studied by equilibrium dialysis and isothermal titration calorimetry (ITC) at 25 degrees C. A concomitant net release of protons with AMP to phosphorylase binding was detected carrying out calorimetric experiments in three buffers having different heats of ionization at 25 degrees C. Four binding sites were found for AMP in the dimeric enzyme, which would correspond to the activator and the inhibitor sites identified in the muscle isozyme. The affinity of AMP for these four sites is similar. Thus, the binding of AMP to the activator sites seems to be non-cooperative and it does not perform the conformational change necessary to activate the enzyme. Moreover, the inhibitor sites are occupied almost in the same extension that the activator sites, which would impair any activation of the enzyme.

The binding of adenosine 5'-monophosphate to liver glycogen phosphorylase a (EC 2.4.1.1) has been studied by size exclusion high performance liquid chromatography and isothermal titration microcalorimetry at pH 6.9 over a temperature range of 25 to 35 degrees C. The results are compared with those of the binding of the same nucleotide to the muscle isozyme and to liver phosphorylase b. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of the nucleotide. The dimer of liver glycogen phosphorylase a has been shown to have two equal and independent sites for 5'-AMP, which would correspond to the activator sites identified in the muscle isozyme. The binding constants as well as the changes in Gibbs energy, enthalpy, and entropy per site for 5'-AMP binding were calculated at each temperature. The results show that the major contribution to the negative value of DeltaG0 stems from the value of DeltaH in the range of 25 to 35 degrees C. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative DeltaCp of binding equal to -1.45 +/- 0.02 kJ K-1 (mol of 5'-AMP bound)-1, which suggests significant changes in the polar and apolar surfaces accessible to the solvent.

The binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) to Lactobacillus casei recombinant thymidylate synthase has been studied by isothermal titration microcalorimetry at pH 7.1 over the temperature range 16-35 degrees C. Calorimetric measurements in various buffer systems with different heats of ionization suggest that a proton uptake is involved in the binding process of the nucleotide. In the temperature range investigated, the mol protons bound/mol nucleotide increases as the temperature decreases. A model of two equal and independent sites fits well with the binding isotherms for thymidylate synthase. The binding constants, the changes in Gibbs energy, enthalpy, and entropy/site for FdUMP binding were calculated at each temperature. The results show that the binding is driven by both enthalpy and entropy contributions in the range 16-35 degrees C. The enthalpy changes become more negative as the temperature increases, with delta Cp = -170 +/- 20 J.K-1.(mol FdUMP bound)-1. The behavior of the system supports the observation that FdUMP binds to thymidylate synthase without producing profound conformational changes in the protein dimer.

The thermal behaviour of bovine-brain myelin membrane has been studied by high-sensitivity differential scanning calorimetry, Fourier-transform infrared spectroscopy and thermal gel analysis. Spectroscopic results indicate that protein transitions take place between 60 degrees C and 90 degrees C, while thermal gel analysis has provided the thermal denaturation profiles of myelin proteolipid, DM-20 protein and the Wolfgram Fraction. An irreversible calorimetric transition centred at 80.3 +/- 0.2 degrees C with a specific enthalpy of 4.7 +/- 0.6 J/g of total protein has been assigned to the thermal denaturation of myelin proteolipid and DM-20 protein. The effects of the myelin storage conditions, scan rate, ionic strength and pH on this calorimetric transition have also been investigated. The thermal transition of the proteolipid practically disappears after treatment of the myelin with different amounts of chloroform-methanol 2:1 (v/v), a treatment which is generally used in proteolipid purification. On the other hand, the addition of several detergents to myelin only causes minor modifications to this transition, which then occurs at about 70 degrees C, with a specific enthalpy of between 2.5 and 3.6 J/g of total protein. These results appear to show that detergents preserve the native conformation of the proteolipid far more than do organic solvents. Hence the use of detergents would seem to be the appropriate method for proteolipid purification.