Volumetric Characterizations of the Native, Molten Globule and Unfolded States of Cytochrome cat Acidic pH

Cytochrome ccan exist in a native (N), a molten globule (MG) or an unfolded (U) state depending on solution conditions. We have used high-precision ultrasonic and densimetric techniques to measure volume and compressibility changes accompanying the N to MG, N to U and U to MG transitions of the protein. For the N to MG transition (induced by lowering the pH to 2 in the presence of 200 mM CsCl), we measure a volume increase of 0.014 cm 3g −1and a compressibility increase of 3.8x10 −6cm 3g −1bar −1. For the N to U transition (induced by lowering the pH to 2 in the absence of salt), we measure a volume increase of 0.010 cm 3g −1and a compressibility decrease of 2.0x10 −6cm 3g −1bar −1. For the U to MG transition at pH 2 (induced by adding CsCl up to 200 mM), we measure a volume increase of 0.006 cm 3g −1and a compressibility increase of 6.8x10 −6cm 3g −1bar −1. We interpret these data to reach the following conclusions about the three states of cytochrome c. (1) A solvent-inaccessible core is preserved in the molten globule state, with the volume of this core being about 40% of the intrinsic volume of native cytochrome c. (2) The coefficient of the adiabatic compressibility of this preserved molten globule core is 61x10 −6bar −1, a value that is over four times higher than that of the interior of the native protein. This result is consistent with the interior of the preserved MG core being liquid-like in contrast to the more tightly packed, solid-like interior of the native state. (3) In the unfolded state of cytochrome c, only 70 to 80% of the surface area of a fully unfolded conformation is exposed to the solvent, a result that reflects some level of order in the ‘`denatured’' state. (4) The relative volume fluctuations of the solvent-inaccessible interiors of the native, molten globule and unfolded states are equal to 0.6%, 2.0% and 2.9%, respectively. These data are consistent with the solvent-inaccessible core of the molten globule state being much more loosely packed than the core of the native state. In fact, the fluctuations in the molten globule and unfolded states are so high that one cannot exclude the possibility that formally buried atomic groups transiently contact solvent molecules. To the best of our knowledge, the data reported here provide the first characterizations of the intrinsic volume and compressibility properties of the native, molten globule and unfolded states of a single protein. We discuss in terms of the current protein literatu