Pressure perturbation calorimetry of unfolded proteins.

We report the application of pressure perturbation calorimetry (PPC) to study unfolded proteins. Using PPC we have measured the temperature dependence of the thermal expansion coefficient, α(T), in the unfolded state of apocytochrome C and reduced BPTI. We have shown that α(T) is a nonlinear function and decreases with increasing temperature. The decrease is most significant in the low (2-55 °C) temperature range. We have also tested an empirical additivity approach to predict α(T) of unfolded state from the amino acid sequence using α(T) values for individual amino acids. A comparison of the experimental and calculated functions shows a very good agreement, both in absolute values of α(T) and in its temperature dependence. Such an agreement suggests the applicability of using empirical calculations to predict α(T) of any unfolded protein.

Modeling the micellization behavior of mixed and pure n-alkyl-maltosides.

The micellization behavior of a series of n-alkyl-maltosides in aqueous solution was studied by isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) at 25 degrees C. Demicellization experiments were conducted with single component micelles of octyl (OM), nonyl (NM), decyl (DM), undecyl (UM), and dodecyl (lauryl, LM) maltoside and binary mixtures of LM with OM, NM, DM and UM, respectively. A model was derived on the basis of the pseudophase approximation to fit the complete demicellization curves. It yielded good global fits of the curves obtained at different mixing ratios and ranging over >3 orders of magnitude in concentrations. It provides a quantitative explanation for the two-range coassociation behavior of the surfactant mixtures also in the absence of second critical micelle concentration (CMC) phenomena. The hydrodynamic radius, RH, of the mixed micelles was the average of that of the pure components as seen by noninvasive backscattering (NIBS) DLS. Methylene group contributions were constant for octyl through myristyl chains, amounting to -3.1 kJ/mol for the standard free energy and -1.8 kJ/mol for the enthalpy of micellization. RH increased by 0.25 nm per methylene.

Monitoring detergent-mediated solubilization and reconstitution of lipid membranes by isothermal titration calorimetry.

The solubilization and reconstitution of biological or liposomal membranes by detergents and biomolecules with detergent-like properties play a major role for technical applications (e.g., the isolation of membrane proteins) and biological phenomena (of, e.g., amphiphilic peptides). It is therefore important to know and understand the amounts of a given detergent required for the onset and completion of membrane solubilization and the detergent-lipid interactions in general. Lipid-detergent systems can form a variety of aggregate structures, which can be grouped into two pseudophases (lamellae and micelles) so that solubilization can be approximately described as a phase transition. Here we present a protocol for establishing the phase diagram and a detailed thermodynamic description of a lipid-detergent system based on isothermal titration calorimetry (ITC). The protocol can also be used to detect additive-induced membrane destabilization, permeabilization, domain formation and lipid-dependent transitions between rod-like and spherical micelles. A minimal protocol consisting of all sample preparation procedures and a single solubilization experiment can be accomplished within 2 days; a more extensive series comprising both solubilization and reconstitution experiments requires several days to a few weeks, depending on the number of titrations performed.

Helix-coil transition of DNA monitored by pressure perturbation calorimetry.

We report the first use of pressure perturbation calorimetry (PPC) to characterize the heat-induced helix-coil transition of DNA polymers. The alternating copolymer poly[d(A-T)] was studied in aqueous solutions containing 5.2 and 18.2 mM Na+; it exhibited helix-coil transition temperatures of 33.6 and 44.7 degrees C, respectively. The transition is accompanied by a negative molar volume change, DeltaV) -2.6 and -2.1 mL/mol (base pair), respectively, and an increase in the coefficient of thermal expansion, Deltaalpha=+5x10(-4) K(-1) (at both ionic strengths). These values are consistent with a greater hydration of the coil form. The larger water-accessible surface area of the coil causes more water molecules to assume a bound, more densely packed structure that then gradually decreases with increasing temperature, leading to a larger value of R. The magnitude of the volume changes detected by PPC were larger than those deduced from high-pressure UV spectroscopy, shedding light on the effect of pressure on DeltaV. The shape of the PPC peak was nearly identical to the shape of the DSC peak, providing direct evidence for the correlation between the molar volume change and enthalpy change for the helix to coil transition of DNA.

Additive action of two or more solutes on lipid membranes.

A wide variety of biological processes, pharmaceutical applications, and technical procedures is based on the combined action of two or more soluble compounds to perturb, permeabilize, or lyse biological membranes. Here we present a general model describing the additive action of solutes on the properties of membranes or micelles. The onset and completion of membrane solubilization induced by two surfactants (lauryl maltoside, with nonyl maltoside, octyl glucoside, or CHAPS, respectively) are very well described by our model on the basis of their individual partition coefficients, cmc's, and critical mole ratios R e sat and R e sol as detected by isothermal titration calorimetry. This suggests that the thermodynamic phase transition is governed by a single parameter (e.g., spontaneous curvature) in spite of the complexity of structural changes. Such surfactant mixtures show unique features such as nonlinear solubilization boundaries and concentration-dependent effective partition coefficients. Other phenomena such as membrane leakage are predicted to obey additive action if the solutes act via the same mechanism (e.g., toroidal pore formation) but deviate from the model in the case of independent, synergistic, or antagonistic action.

Uptake and release protocol for assessing membrane binding and permeation by way of isothermal titration calorimetry.

The activity of many biomolecules and drugs crucially depends on whether they bind to biological membranes and whether they translocate to the opposite lipid leaflet and trans aqueous compartment. A general strategy to measure membrane binding and permeation is the uptake and release assay, which compares two apparent equilibrium situations established either by the addition or by the extraction of the solute of interest. Only solutes that permeate the membrane sufficiently fast do not show any dependence on the history of sample preparation. This strategy can be pursued for virtually all membrane-binding solutes, using any method suitable for detecting binding. Here, we present in detail one example that is particularly well developed, namely the nonspecific membrane partitioning and flip-flop of small, nonionic solutes as characterized by isothermal titration calorimetry. A complete set of experiments, including all sample preparation procedures, can typically be accomplished within 2 days. Analogous protocols for studying charged solutes, virtually water-insoluble, hydrophobic compounds or specific ligands are also considered.