Thermodynamics of ribonuclease T1 denaturation.


Abstract

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves. Study holds ProTherm entries: 3938, 3939, 3940, 3941, 3942, 3943, 3944, 3945, 3946, 3947, 3948, 3949, 3950, 3951, 3952, 3953, 3954, 3955, 3956, 3957, 10591, 10592, 10593, 10594, 10595, 10596, 10597, 10598, 10599, 10600, 10601, 10602, 10603, 10604, 10605, 10606, 10607, 10608, 10609 Extra Details: thermodynamic parameters; heat capacity change; preferential,binding of cations; folded conformation

Submission Details

ID: 4WUXubPN

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Hu CQ;Sturtevant JM;Thomson JA;Erickson RE;Pace CN,Biochemistry (1992) Thermodynamics of ribonuclease T1 denaturation. PMID:1591247
Additional Information

Study Summary

Number of data points 146
Proteins Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp ionic:MgCl2: 0.80 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal ionic:MgCl2: 0.80 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm ionic:MgCl2: 0.80 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH ionic:MgCl2: 0.80 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp ionic:MgCl2: 0.60 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal ionic:MgCl2: 0.60 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm ionic:MgCl2: 0.60 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH ionic:MgCl2: 0.60 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp ionic:MgCl2: 0.40 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal ionic:MgCl2: 0.40 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm ionic:MgCl2: 0.40 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH ionic:MgCl2: 0.40 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp buffers:PIPES: 30 mM, ionic:MgCl2: 0.20 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal buffers:PIPES: 30 mM, ionic:MgCl2: 0.20 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm buffers:PIPES: 30 mM, ionic:MgCl2: 0.20 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH buffers:PIPES: 30 mM, ionic:MgCl2: 0.20 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp buffers:PIPES: 30 mM, ionic:MgCl2: 0.10 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal buffers:PIPES: 30 mM, ionic:MgCl2: 0.10 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm buffers:PIPES: 30 mM, ionic:MgCl2: 0.10 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH buffers:PIPES: 30 mM, ionic:MgCl2: 0.10 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp ionic:MgCl2: 0.06 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal ionic:MgCl2: 0.06 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm ionic:MgCl2: 0.06 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH ionic:MgCl2: 0.06 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp ionic:MgCl2: 0.03 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal ionic:MgCl2: 0.03 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm ionic:MgCl2: 0.03 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH ionic:MgCl2: 0.03 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp buffers:PIPES: 30 mM, ionic:MgCl2: 0.02 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal buffers:PIPES: 30 mM, ionic:MgCl2: 0.02 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm buffers:PIPES: 30 mM, ionic:MgCl2: 0.02 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH buffers:PIPES: 30 mM, ionic:MgCl2: 0.02 M, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp ionic:MgCl2: 0.01 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal ionic:MgCl2: 0.01 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm ionic:MgCl2: 0.01 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH ionic:MgCl2: 0.01 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp ionic:MgCl2: 0 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHcal ionic:MgCl2: 0 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: Tm ionic:MgCl2: 0 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dHvH ionic:MgCl2: 0 M, buffers:PIPES: 30 mM, prot_conc:1.06-1.56 mg/mL, pH:7.0 ; Experimental Assay: dCp prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 1.60 M, pH:7.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 1.60 M, pH:7.0 ; Experimental Assay: Tm prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 1.60 M, pH:7.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 1.60 M, pH:7.0 ; Experimental Assay: dCp prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 1.20 M, pH:7.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 1.20 M, pH:7.0 ; Experimental Assay: Tm prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 1.20 M, pH:7.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 1.20 M, pH:7.0 ; Experimental Assay: dCp prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.80 M, pH:7.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.80 M, pH:7.0 ; Experimental Assay: Tm prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.80 M, pH:7.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.80 M, pH:7.0 ; Experimental Assay: dCp prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.60 M, pH:7.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.60 M, pH:7.0 ; Experimental Assay: Tm prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.60 M, pH:7.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.60 M, pH:7.0 ; Experimental Assay: dCp ionic:NaCl: 0.40 M, prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, pH:7.0 ; Experimental Assay: dHcal ionic:NaCl: 0.40 M, prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, pH:7.0 ; Experimental Assay: Tm ionic:NaCl: 0.40 M, prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, pH:7.0 ; Experimental Assay: dHvH ionic:NaCl: 0.40 M, prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, pH:7.0 ; Experimental Assay: dCp prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.20 M, pH:7.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.20 M, pH:7.0 ; Experimental Assay: Tm prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.20 M, pH:7.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.20 M, pH:7.0 ; Experimental Assay: dCp prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.10 M, pH:7.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.10 M, pH:7.0 ; Experimental Assay: Tm prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.10 M, pH:7.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.10 M, pH:7.0 ; Experimental Assay: dCp prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.05 M, pH:7.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.05 M, pH:7.0 ; Experimental Assay: Tm prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.05 M, pH:7.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, ionic:NaCl: 0.05 M, pH:7.0 ; Experimental Assay: dCp ionic:NaCl: 0 M, prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, pH:7.0 ; Experimental Assay: dHcal ionic:NaCl: 0 M, prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, pH:7.0 ; Experimental Assay: Tm ionic:NaCl: 0 M, prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, pH:7.0 ; Experimental Assay: dHvH ionic:NaCl: 0 M, prot_conc:2.63 mg/mL, buffers:PIPES: 30 mM, pH:7.0 ; Experimental Assay: Cm temp:40.1 C ; Experimental Assay: m temp:40.1 C ; Experimental Assay: dG_H2O temp:40.1 C ; Experimental Assay: Cm temp:34.75 C ; Experimental Assay: m temp:34.75 C ; Experimental Assay: dG_H2O temp:34.75 C ; Experimental Assay: Cm temp:29.0 C ; Experimental Assay: m temp:29.0 C ; Experimental Assay: dG_H2O temp:29.0 C ; Experimental Assay: Cm temp:27.0 C ; Experimental Assay: m temp:27.0 C ; Experimental Assay: dG_H2O temp:27.0 C ; Experimental Assay: Cm temp:25.0 C ; Experimental Assay: m temp:25.0 C ; Experimental Assay: dG_H2O temp:25.0 C ; Experimental Assay: Cm temp:23.05 C ; Experimental Assay: m temp:23.05 C ; Experimental Assay: dG_H2O temp:23.05 C ; Experimental Assay: Cm temp:21.1 C ; Experimental Assay: m temp:21.1 C ; Experimental Assay: dG_H2O temp:21.1 C ; Experimental Assay: Cm temp:19.2 C ; Experimental Assay: m temp:19.2 C ; Experimental Assay: dG_H2O temp:19.2 C ; Experimental Assay: Cm temp:14.9 C ; Experimental Assay: m temp:14.9 C ; Experimental Assay: dG_H2O temp:14.9 C ; Experimental Assay: Cm temp:9.98 C ; Experimental Assay: m temp:9.98 C ; Experimental Assay: dG_H2O temp:9.98 C ; Experimental Assay: dCp prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:2.2 ; Experimental Assay: dHcal prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:2.2 ; Experimental Assay: Tm prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:2.2 ; Experimental Assay: dHvH prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:2.2 ; Experimental Assay: dCp pH:2.8, prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: ; Experimental Assay: dHcal pH:2.8, prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: ; Experimental Assay: Tm pH:2.8, prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: ; Experimental Assay: dHvH ionic:: , prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, pH:2.8 ; Experimental Assay: dCp prot_conc:2.63 mg/ml, buffers:acetate: 30 mM, ionic:: , pH:4.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/ml, buffers:acetate: 30 mM, ionic:: , pH:4.0 ; Experimental Assay: Tm prot_conc:2.63 mg/ml, buffers:acetate: 30 mM, ionic:: , pH:4.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/ml, buffers:acetate: 30 mM, ionic:: , pH:4.0 ; Experimental Assay: dCp pH:5.0, prot_conc:2.63 mg/ml, buffers:acetate: 30 mM, ionic:: ; Experimental Assay: dHcal pH:5.0, prot_conc:2.63 mg/ml, buffers:acetate: 30 mM, ionic:: ; Experimental Assay: Tm pH:5.0, prot_conc:2.63 mg/ml, buffers:acetate: 30 mM, ionic:: ; Experimental Assay: dHvH pH:5.0, prot_conc:2.63 mg/ml, buffers:acetate: 30 mM, ionic:: ; Experimental Assay: dCp pH:6.0, prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: ; Experimental Assay: dHcal pH:6.0, prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: ; Experimental Assay: Tm pH:6.0, prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: ; Experimental Assay: dHvH pH:6.0, prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: ; Experimental Assay: dCp prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: , pH:7.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: , pH:7.0 ; Experimental Assay: Tm prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: , pH:7.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: , pH:7.0 ; Experimental Assay: dCp pH:7.5, prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: ; Experimental Assay: dHcal pH:7.5, prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: ; Experimental Assay: Tm pH:7.5, prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: ; Experimental Assay: dHvH pH:7.5, prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: ; Experimental Assay: dCp prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: , pH:8.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: , pH:8.0 ; Experimental Assay: Tm prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: , pH:8.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/ml, buffers:PIPES: 30 mM, ionic:: , pH:8.0 ; Experimental Assay: dCp prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:9.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:9.0 ; Experimental Assay: Tm prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:9.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:9.0 ; Experimental Assay: dCp prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:10.0 ; Experimental Assay: dHcal prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:10.0 ; Experimental Assay: Tm prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:10.0 ; Experimental Assay: dHvH prot_conc:2.63 mg/ml, buffers:glycine: 30 mM, ionic:: , pH:10.0
Libraries Mutations for sequence ACDYTCGSNCYSSSDVSTAQAAGYQLHEDGETVGSNSYPHKYNNYEGFDFSVSSPYYEWPILSSGDVYSGGSPGADRVVFNENNQLAGVITHTGASGNNFVECT

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
1B2M 1998-11-27T00:00:00+0000 2.0 THREE-DIMENSIONAL STRUCTURE OF RIBONULCEASE T1 COMPLEXED WITH AN ISOSTERIC PHOSPHONATE ANALOGUE OF GPU: ALTERNATE SUBSTRATE BINDING MODES AND CATALYSIS.
1BIR 1996-01-04T00:00:00+0000 1.8 RIBONUCLEASE T1, PHE 100 TO ALA MUTANT COMPLEXED WITH 2' GMP
1BU4 1998-09-11T00:00:00+0000 1.9 RIBONUCLEASE 1 COMPLEX WITH 2'GMP
1BVI 1998-09-15T00:00:00+0000 1.9 RIBONUCLEASE T1 (WILDTYPE) COMPLEXED WITH 2'GMP
1CH0 1999-03-30T00:00:00+0000 2.3 RNASE T1 VARIANT WITH ALTERED GUANINE BINDING SEGMENT
1DET 1996-02-20T00:00:00+0000 1.8 RIBONUCLEASE T1 CARBOXYMETHYLATED AT GLU 58 IN COMPLEX WITH 2'GMP
1FYS 2000-10-03T00:00:00+0000 2.0 Ribonuclease T1 V16C mutant
1FZU 2000-10-04T00:00:00+0000 1.8 RNAse T1 V78A mutant
1G02 2000-10-05T00:00:00+0000 1.86 Ribonuclease T1 V16S mutant
1GSP 1997-11-28T00:00:00+0000 2.2 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS, 1 DAY

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Guanyl-specific ribonuclease T1 P00651 RNT1_ASPOR