title: A new alternative method to quantify residual structure in 'unfolded' proteins.
abstract: Pig (pCSD1) and human (hCSD1) calpastatin domain 1 proteins were studied to characterize common features of the denatured state of proteins. These proteins were chosen for the present investigation, because pCSD1 was suggested previously to be unstructured in water even at 25 degrees C (1) [T. Konno et al., Biochim. Biophys. Acta 1342 (1997) 73-82]. hCSD1 could be expected to exhibit similar features on the basis of preliminary spectroscopic studies. In the present study, the experimental grounds for the estimate of residual structure in the unfolded state were differential scanning calorimetry heat capacity and circular dichroism (CD) measurements over the temperature range 10-80 degrees C. At selected temperatures, we studied also the effect of guanidinium hydrochloride (GdnHCl) which is known to promote further unfolding of the polypeptide chain. All other measurements were performed at pH 6 in pure water. The present results support the conclusion that the comparison of the experimentally obtained heat capacity data with theoretical heat capacity values calculated on the basis of a newly established increment system gives insight into the degree of hydration of the unfolded polypeptide chain. The percentage by which the experimental heat capacity of the unfolded polypeptide chain differs from the calculated heat capacity permits a quantitative estimate of the residual structure. This estimate is in good agreement with that based on CD absorption. The heat capacity approach has the advantage of comparing fully hydrated and partially hydrated residues in the same aqueous environment, whereas for example spectroscopic measurements, such as CD, are generally referred to the fully unfolded chain in concentrated urea or GdnHCl solutions. As the unfolded chains of pCSD1 and hCSD1 exhibit a smaller heat capacity than that calculated on the new peptide-based increment system [M. Häckel et al., J. Mol. Biol. 291 (1999) 197-213], we conclude that the residues in the unfolded polypeptide chain are less hydrated than the same residues in oligopeptides. This suboptimal hydration is the result of residual structure in the chain as observed in both CD and heat capacity measurements.
authors.affiliation: Institut für Physikalische Chemie der Westfälischen Wilhelms-Universität Münster, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Häckel
authors.forename: T
authors.initials: T
authors.lastname: Konno
authors.forename: H
authors.initials: H
authors.lastname: Hinz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013552:Swine
chemical_list: D002135:Calcium-Binding Proteins; C033668:calpastatin