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 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