4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization.


Abstract

The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9. Study holds ProTherm entries: 17040, 17041, 17042, 17043, 17044, 17045, 17046, 17047, 17048, 17049, 17050, 17051, 17052, 17053, 17054, 17055, 17056, 17057, 17058 Extra Details: Scan rate is 0.5 K/min. ribonuclease A; 4-chlorobutan-1-ol; thermal denaturation; differential scanning calorimetry; enthalpy; activation energy

Submission Details

ID: iyFQGCwR4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Mehta R;Kundu A;Kishore N,Int. J. Biol. Macromol. (2004) 4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization. PMID:15178004
Additional Information

Study Summary

Number of data points 45
Proteins Ribonuclease pancreatic ; Ribonuclease pancreatic
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dHcal prot_conc:0.44 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: Tm prot_conc:0.44 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: dHcal prot_conc:0.37 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: Tm prot_conc:0.37 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: dHcal prot_conc:0.29 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: Tm prot_conc:0.29 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: dHcal prot_conc:0.22 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: Tm prot_conc:0.22 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: dHcal prot_conc:0.15 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: Tm prot_conc:0.15 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: dHcal prot_conc:0.07 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: Tm prot_conc:0.07 mM, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: dHcal prot_conc:0.00, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: Tm prot_conc:0.00, details:Additives 4-chlorobutan-1-ol (100 mM) ; Experimental Assay: dCp details:Additives 4-chlorobutan-1-ol (0.400 M) ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.400 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.400 M) ; Experimental Assay: dCp details:Additives 4-chlorobutan-1-ol (0.250 M) ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.250 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.250 M) ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.200 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.200 M) ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.175 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.175 M) ; Experimental Assay: dHcal details:Additives 4-chlorobutan-1-ol (0.150 M), prot_conc:0.32 mM ; Experimental Assay: Tm details:Additives 4-chlorobutan-1-ol (0.150 M), prot_conc:0.32 mM ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.125 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.125 M) ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.100 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.100 M) ; Experimental Assay: dCp details:Additives 4-chlorobutan-1-ol (0.050 M) ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.050 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.050 M) ; Experimental Assay: dCp details:Additives 4-chlorobutan-1-ol (0.020 M) ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.020 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.020 M) ; Experimental Assay: dCp details:Additives 4-chlorobutan-1-ol (0.010 M) ; Experimental Assay: dHcal details:Additives 4-chlorobutan-1-ol (0.010 M), prot_conc:0.32 mM ; Experimental Assay: Tm details:Additives 4-chlorobutan-1-ol (0.010 M), prot_conc:0.32 mM ; Experimental Assay: dCp details:Additives 4-chlorobutan-1-ol (0.000 M) ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.000 M) ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives 4-chlorobutan-1-ol (0.000 M) ; Experimental Assay: dCp details:Additives ; Experimental Assay: dHcal prot_conc:0.32 mM, details:Additives ; Experimental Assay: Tm prot_conc:0.32 mM, details:Additives
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
6XHD 2020-06-18T00:00:00+0000 1.51 Structure of Prolinyl-5'-O-adenosine phosphoramidate
6XHE 2020-06-18T00:00:00+0000 1.88 Structure of beta-prolinyl 5'-O-adenosine phosphoramidate
3QL2 2011-02-02T00:00:00+0000 1.49 Crystal Structure of Ribonuclease A Variant A4C/D83E/V118C
1YMW 2005-01-21T00:00:00+0000 1.5 The study of reductive unfolding pathways of RNase A (Y92G mutant)
4G90 2012-07-23T00:00:00+0000 1.9 Crystal structure of Ribonuclease A in complex with 5e
3D6P 2008-05-20T00:00:00+0000 1.6 RNase A- 5'-Deoxy-5'-N-morpholinouridine complex
3D6Q 2008-05-20T00:00:00+0000 1.6 The RNase A- 5'-Deoxy-5'-N-piperidinouridine complex
1KH8 2001-11-29T00:00:00+0000 2.0 Structure of a cis-proline (P114) to glycine variant of Ribonuclease A
4WYP 2014-11-17T00:00:00+0000 1.5 The crystal structure of the A109G mutant of RNase A in complex with 5'AMP

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