Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study.


Abstract

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods. Study holds ProTherm entries: 5459, 5460, 5461, 5462, 5463, 5464, 5465, 5466, 5467, 5468, 5469, 5470 Extra Details: thermal and solvent denaturation; UV spectroscopy

Submission Details

ID: soPpY7953

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Poklar N;Petrovcic N;Oblak M;Vesnaver G,Protein Sci. (1999) Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study. PMID:10211829
Additional Information

Study Summary

Number of data points 24
Proteins Ribonuclease pancreatic ; Ribonuclease pancreatic
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Tm buffers:None: -, ionic:: , pH:7.0 ; Experimental Assay: dHcal buffers:None: -, ionic:: , pH:7.0 ; Experimental Assay: Tm ionic:: , buffers:None: -, pH:7.0 ; Experimental Assay: dHvH buffers:None: -, ionic:: , pH:7.0 ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:3.5 ; Experimental Assay: dHcal buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:3.5 ; Experimental Assay: Tm pH:3.5, buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M ; Experimental Assay: dHvH buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:3.5 ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, pH:3.0, ionic:NaCl: 0.1 M ; Experimental Assay: dHcal buffers:glycine-HCl: 0.1 M/0.1 M, pH:3.0, ionic:NaCl: 0.1 M ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:3.0 ; Experimental Assay: dHvH buffers:glycine-HCl: 0.1 M/0.1 M, pH:3.0, ionic:NaCl: 0.1 M ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:2.0 ; Experimental Assay: dHcal buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:2.0 ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:2.0 ; Experimental Assay: dHvH buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:2.0 ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:1.5 ; Experimental Assay: dHcal buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:1.5 ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:1.5 ; Experimental Assay: dHvH buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:1.5 ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:1.1 ; Experimental Assay: dHcal pH:1.1, buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M ; Experimental Assay: Tm buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M, pH:1.1 ; Experimental Assay: dHvH pH:1.1, buffers:glycine-HCl: 0.1 M/0.1 M, ionic:NaCl: 0.1 M
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
1A2W 1998-01-12T00:00:00+0000 2.1 CRYSTAL STRUCTURE OF A 3D DOMAIN-SWAPPED DIMER OF BOVINE PANCREATIC RIBONUCLEASE A
1A5P 1998-02-17T00:00:00+0000 1.6 C[40,95]A VARIANT OF BOVINE PANCREATIC RIBONUCLEASE A
1A5Q 1998-02-17T00:00:00+0000 2.3 P93A VARIANT OF BOVINE PANCREATIC RIBONUCLEASE A
1AFK 1997-03-08T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF RIBONUCLEASE A IN COMPLEX WITH 5'-DIPHOSPHOADENOSINE-3'-PHOSPHATE
1AFL 1997-03-08T00:00:00+0000 1.7 RIBONUCLEASE A IN COMPLEX WITH 5'-DIPHOSPHOADENOSINE 2'-PHOSPHATE AT 1.7 ANGSTROM RESOLUTION
1AFU 1997-03-14T00:00:00+0000 2.0 STRUCTURE OF RIBONUCLEASE A AT 2.0 ANGSTROMS FROM MONOCLINIC CRYSTALS
1AQP 1997-07-31T00:00:00+0000 2.0 RIBONUCLEASE A COPPER COMPLEX
1B6V 1999-01-18T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF A HYBRID BETWEEN RIBONUCLEASE A AND BOVINE SEMINAL RIBONUCLEASE
1BEL 1995-12-21T00:00:00+0000 1.6 HYDROLASE PHOSPHORIC DIESTER, RNA

Relevant UniProtKB Entries

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