Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A.
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 overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed 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 enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.
Catanzano
F
F
Department of Chemistry, University of Napoli Federico II Via Mezzocannone, Italy.
Graziano
G
G
Capasso
S
S
Barone
G
G
D000577:Amides / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003568:Cytidine Monophosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D000577:Amides; D001216:Asparagine; D012259:Ribonuclease, Pancreatic; D003568:Cytidine Monophosphate