Abstract:
:We report the first protein phase-diagram characterized by a combination of volumetric, calorimetric, and spectroscopic techniques. More specifically, we use ultrasonic velocimetry, densimetry, and differential scanning calorimetry, in conjunction with UV absorbance and CD spectroscopy 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 protein remains in the native state over the entire pH range investigated (pH 1 to 10). The melting profiles of the native state reveal three distinct pH domains in which protein denaturation produces different final states. Below pH 3.1, we find the heat-induced denatured state of the 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, while being structurally similar to the urea-unfolded state. At intermediate pH values (between pH 3.1 and 7), we find the heat-induced denatured state to exhibit properties characteristic of both the MG and U states. Although at room temperature the protein remains native within the whole pH range studied (pH 1 to 10), our volumetric data reveal that the native state slightly "softens" at low pH, probably, due to pH-induced alterations in electrostatic forces causing the packing of the protein interior at low pH and room temperature to become less "tight". This softening of the protein at low pH is reflected in an 8% increase in the intrinsic compressibility, kM, of the protein "native" 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 core of the native state. By contrast, our volumetric data are consistent 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.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Chalikian TV,Völker J,Anafi D,Breslauer KJdoi
10.1006/jmbi.1997.1394subject
Has Abstractpub_date
1997-11-28 00:00:00pages
237-52issue
2eissn
0022-2836issn
1089-8638pii
S0022-2836(97)91394-2journal_volume
274pub_type
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