THE NATIVE AND THE HEAT-INDUCED DENATURED STATES OF ALPHA-CHYMOTRYPSINOGEN-A - THERMODYNAMIC AND SPECTROSCOPIC STUDIES

We report the first protein phase-diagram characterized by a combinati on of volumetric, calorimetric, and spectroscopic techniques. More spe cifically, we use ultrasonic velocimetry, densimetry, and differential scanning calorimetry, in conjunction with UV absorbance and CD spectr oscopy to detect and to characterize the conformational transitions of alpha-chymotrypsinogen A as a function of both pH and temperature. As judged by the CD spectra, we find that, at room temperature, the prot ein remains in the native state over the entire pH range investigated (pH 1 to 10). The melting profiles of the native state reveal three di stinct pH domains in which protein denaturation produces different fin al states. Below pH 3.1, we find the heat-induced denatured state of t he protein to be molten globule (MG), lacking the native-like tertiary structure, while exhibiting significant secondary structural elements . At neutral and alkaline pH, we find the heat-induced denatured state to be unfolded (U), lacking both tertiary and secondary structures, w hile being structurally similar to the urea-unfolded state. At interme diate pH values (between pH 3.1 and 7), we find the heat-induced denat ured state to exhibit properties characteristic of both the MG and U s tates. Although at room temperature the protein remains native within the whole pH range studied (pH 1 to 10), our volumetric data reveal th at the native state slightly ''softens'' at low pH, probably, due to p H-induced alterations in electrostatic forces causing the packing of t he protein interior at low pH and room temperature to become less ''ti ght''. This softening of the protein at low pH is reflected in an 8% i ncrease in the intrinsic compressibility, k(M), of the protein ''nativ e'' state. Our volumetric data also allow us to conclude that the heat -induced MG state retains a liquid-like, water-inaccessible core, with a volume that corresponds to about 40% of the solvent-inaccessible co re of the native state. By contrast, our volumetric data are consisten t with the U state of the protein being essentially unfolded, with the majority of its constituent atomic groups being solvent exposed and, therefore, strongly hydrated. (C) 1997 Academic Press Limited.