THERMODYNAMIC ANALYSIS OF THE EFFECT OF SELECTIVE MONODEAMIDATION AT ASPARAGINE-67 IN RIBONUCLEASE-A

Citation
F. Catanzano et al., THERMODYNAMIC ANALYSIS OF THE EFFECT OF SELECTIVE MONODEAMIDATION AT ASPARAGINE-67 IN RIBONUCLEASE-A, Protein science, 6(8), 1997, pp. 1682-1693
Citations number
71
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0961-8368
Volume
6
Issue
8
Year of publication
1997
Pages
1682 - 1693
Database
ISI
SICI code
0961-8368(1997)6:8<1682:TAOTEO>2.0.ZU;2-P
Abstract
Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overal l stability of the protein, In order to evaluate the thermodynamic eff ects of this reaction in proteins, we investigated the temperature-ind uced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl res idue, as a consequence of an in vitro deamidation reaction, Differenti al scanning calorimetry measurements were performed in the pH range 3. 0-6.0 where the unfolding process is reversible, according to the rehe ating criterion used, It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In par ticular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 6 5 kJ mol(-1) of denaturation enthalpy at pH 5.0. These results are dis cussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to en thalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entrop ic effects. Finally, the influence of cytidino-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explain ed in terms of its binding on the active site of ribonucleases. The an alysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.