Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A.


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 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. Study holds ProTherm entries: 3528, 3529, 3530, 3531, 3532, 3533, 3534, 3535, 3536, 3537, 3538, 3539, 3540, 3541 Extra Details: protein deamidation; ribonuclease A; thermodynamic stability

Submission Details

ID: SDLXprBt3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:21 p.m.

Version: 1

Publication Details
Catanzano F;Graziano G;Capasso S;Barone G,Protein Sci. (1997) Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A. PMID:9260280
Additional Information

Study Summary

Number of data points 49
Proteins Ribonuclease pancreatic ; Ribonuclease pancreatic
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: dCp buffers:HEPPSO: 0.1 M, pH:8.5 ; Experimental Assay: dHcal buffers:HEPPSO: 0.1 M, pH:8.5 ; Experimental Assay: Tm buffers:HEPPSO: 0.1 M, pH:8.5 ; Experimental Assay: dCp pH:6.0, buffers:MES: 0.1 M ; Experimental Assay: dHcal pH:6.0, buffers:MES: 0.1 M ; Experimental Assay: Tm pH:6.0, buffers:MES: 0.1 M ; Experimental Assay: dCp pH:5.0, buffers:Acetic acid-Sodium acetate: 0.1 M ; Experimental Assay: dHcal pH:5.0, buffers:Acetic acid-Sodium acetate: 0.1 M ; Experimental Assay: Tm pH:5.0, buffers:Acetic acid-Sodium acetate: 0.1 M ; Experimental Assay: dCp pH:4.5, buffers:Acetic acid-Sodium acetate: 0.1 M ; Experimental Assay: dHcal pH:4.5, buffers:Acetic acid-Sodium acetate: 0.1 M ; Experimental Assay: Tm pH:4.5, buffers:Acetic acid-Sodium acetate: 0.1 M ; Experimental Assay: dCp buffers:Acetic acid-Sodium acetate: 0.1 M, pH:4.0 ; Experimental Assay: dHcal buffers:Acetic acid-Sodium acetate: 0.1 M, pH:4.0 ; Experimental Assay: Tm buffers:Acetic acid-Sodium acetate: 0.1 M, pH:4.0 ; Experimental Assay: dCp buffers:glycine-HCl: 0.1 M, pH:3.5 ; Experimental Assay: dHcal buffers:glycine-HCl: 0.1 M, pH:3.5 ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M, pH:3.5 ; Experimental Assay: dCp buffers:glycine-HCl: 0.1 M, pH:3.0 ; Experimental Assay: dHcal buffers:glycine-HCl: 0.1 M, pH:3.0 ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M, pH:3.0 ; Derived Quantity: dTm buffers:HEPPSO: 0.1 M, pH:8.5 ; Derived Quantity: dTm pH:6.0, buffers:MES: 0.1 M ; Derived Quantity: dTm pH:5.0, buffers:Acetic acid-Sodium acetate: 0.1 M ; Derived Quantity: dTm pH:4.5, buffers:Acetic acid-Sodium acetate: 0.1 M ; Derived Quantity: dTm buffers:Acetic acid-Sodium acetate: 0.1 M, pH:4.0 ; Derived Quantity: dTm buffers:glycine-HCl: 0.1 M, pH:3.5 ; Derived Quantity: dTm buffers:glycine-HCl: 0.1 M, pH:3.0
Libraries Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
4WYN 2014-11-17T00:00:00+0000 1.8 The crystal structure of the A109G mutant of RNase A
6XHF 2020-06-18T00:00:00+0000 1.45 Structure of Phenylalanyl-5'-O-adenosine phosphoramidate
6XHE 2020-06-18T00:00:00+0000 1.88 Structure of beta-prolinyl 5'-O-adenosine phosphoramidate
6XHD 2020-06-18T00:00:00+0000 1.51 Structure of Prolinyl-5'-O-adenosine phosphoramidate
1SSC 1994-10-05T00:00:00+0000 2.0 THE 1.6 ANGSTROMS STRUCTURE OF A SEMISYNTHETIC RIBONUCLEASE CRYSTALLIZED FROM AQUEOUS ETHANOL. COMPARISON WITH CRYSTALS FROM SALT SOLUTIONS AND WITH RNASE A FROM AQUEOUS ALCOHOL SOLUTIONS
1SSC 1994-10-05T00:00:00+0000 2.0 THE 1.6 ANGSTROMS STRUCTURE OF A SEMISYNTHETIC RIBONUCLEASE CRYSTALLIZED FROM AQUEOUS ETHANOL. COMPARISON WITH CRYSTALS FROM SALT SOLUTIONS AND WITH RNASE A FROM AQUEOUS ALCOHOL SOLUTIONS
5JMG 2016-04-29T00:00:00+0000 1.85 X-ray structure of the complex between bovine pancreatic ribonuclease and pentachlorocarbonyliridate(III) (4 days of soaking)
6XHC 2020-06-18T00:00:00+0000 1.6 Structure of glycinyl 5'-O-adenosine phosphoramidate
1UN5 2003-09-04T00:00:00+0000 2.6 ARH-II, AN ANGIOGENIN/RNASE A CHIMERA

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI