A new alternative method to quantify residual structure in 'unfolded' proteins

Pig (pCSD1) and human (hCSD1) calpastatin domain 1 proteins were studied to characterize common features of the denatured state of proteins. These pro teins were chosen for the present investigation, because pCSD1 was suggeste d 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 stud ies. In the present study, the experimental grounds for the estimate of res idual structure in the unfolded state were differential scanning calorimetr y heat capacity and circular dichroism (CD) measurements over the temperatu re range 10-80 degrees C. At selected temperatures, we studied also the eff ect 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 theoretic al heat capacity values calculated on the basis of a newly established incr ement system gives insight into the degree of hydration of the unfolded pol ypeptide chain. The percentage by which the experimental heat capacity of t he unfolded polypeptide chain differs from the calculated heat capacity per mits a quantitative estimate of the residual structure. This estimate is in good agreement with that based on CD absorption. The heat capacity approac h has the advantage of comparing fully hydrated and partially hydrated resi dues in the same aqueous environment, whereas for example spectroscopic mea surements, such as CD, are generally referred to the fully unfolded chain i n concentrated urea or GdnHCl solutions. As the unfolded chains of pCSD1 an d hCSD1 exhibit a smaller heat capacity than that calculated on the new pep tide-based increment system [M. Hackel et al., J. Mel. 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 hydr ation is the result of residual structure in the chain as observed in both CD and heat capacity measurements. (C) 2000 Elsevier Science B.V. All right s reserved.