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A structural parameterization of the folding energetics has been used to predict the effect of single amino acid mutations at exposed locations in alpha-helices. The results have been used to derive a structure-based thermodynamic scale of alpha-helix propensities for amino acids. The structure-based thermodynamic analysis was performed for four different systems for which structural and experimental thermodynamic data are available: T4 lysozyme [Blaber et al (1994) J. Mol. Biol.235, 600-624], barnase [Horovitz et al. (1992) J.Mol.Biol.227,560-568], a synthetic leucine zipper [O'Neil & Degrado (1990) Science 250, 646-651], and a synthetic peptide [Lyu et al. (1990) Science 250, 669-673]. These studies have permitted the optimization of the set of solvent-accessible surface areas (ASA) for all amino acids in the unfolded state. It is shown that a single set of structure/thermodynamic parameters accounts well for all the experimental data sets of helix propensities. For T4 lysozyme, the average value of the absolute difference between predicted and experimental delta G values is 0.09 kcal/mol, for barnase 0.14 kcal/mol, for the synthetic coiled-coil 0.11 kcal/mol, and for the synthetic peptide 0.08 kcal/mol. In addition, this approach predicts well the overall stability of the proteins and rationalizes the differences in alpha-helix propensities between amino acids. The excellent agreement observed between predicted and experimental delta G values for all amino acids validates the use of this structural parameterization in free energy calculations for folding or binding.

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The interactions of the targeting sequence of the mitochondrial enzyme ornithine transcarbamylase with phospholipid bilayers of different molecular compositions have been studied by high-sensitivity heating and cooling differential scanning calorimetry, high-sensitivity isothermal titration calorimetry, fluorescence spectroscopy, and electron microscopy. These studies indicate that the leader peptide interacts strongly with dipalmitoylphosphatidylcholine (DPPC) bilayer membranes containing small mole percents of the anionic phospholipids dipalmitoylphosphatidylglycerol (DPPG) or brain phosphatidylserine (brain PS) but not with pure phosphatidylcholines. For the first time, the energetics of the leader peptide-membrane interaction have been measured directly by using calorimetric techniques. At 20 degrees C, the association of the peptide with the membrane is exothermic and characterized by an association constant of 2.3 X 10(6) M-1 in the case of phosphatidylglycerol-containing and 0.35 X 10(6) M-1 in the case of phosphatidylserine-containing phospholipid bilayers. In both cases, the enthalpy of association is -60 kcal/mol of peptide. Additional experiments using fluorescence techniques suggest that the peptide does not penetrate deeply into the hydrophobic core of the membrane. The addition of the leader peptide to DPPC/DPPG (5:1) or DPPC/brain PS (5:1) small sonicated vesicles results in vesicle fusion. The fusion process is dependent on peptide concentration and is maximal at the phase transition temperature of the vesicles and minimal at temperatures below the phase transition.

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The folding-unfolding transition of Fe(III) cytochrome c has been studied with the new technique of multifrequency calorimetry. Multifrequency calorimetry is aimed at measuring directly the dynamics of the energetic events that take place during a thermally induced transition by measuring the frequency dispersion of the heat capacity. This is done by modulating the folding/unfolding equilibrium using a variable frequency, small oscillatory temperature perturbation (approximately 0.05-0.1 degrees C) centered at the equilibrium temperature of the system. Fe(III) cytochrome c at pH 4 undergoes a fully reversible folding/unfolding transition centered at 67.7 degrees C and characterized by an enthalpy change of 81 kcal/mol and heat capacity difference between unfolded and folded states of 0.9 kcal/K*mol. By measuring the temperature dependence of the frequency dispersion of the heat capacity in the frequency range of 0.1-1 Hz it has been possible to examine the time regime of the enthalpic events associated with the transition. The multifrequency calorimetry results indicate that approximately 85% of the excess heat capacity associated with the folding/unfolding transition relaxes with a single relaxation time of 326 +/- 68 ms at the midpoint of the transition region. This is the first time that the time regime in which heat is absorbed and released during protein folding/unfolding has been measured.