pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1.


Abstract

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein. Study holds ProTherm entries: 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310, 2311 Extra Details: Ribonuclease A; conformational stability; electrostatic interactions

Submission Details

ID: zYwYcLvw3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:18 p.m.

Version: 1

Publication Details
Pace CN;Laurents DV;Thomson JA,Biochemistry (1990) pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1. PMID:2110472
Additional Information

Study Summary

Number of data points 24
Proteins Ribonuclease pancreatic ; Ribonuclease pancreatic ; Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: dG_H2O pH:9.91, buffers:glycine: 30 mM ; Experimental Assay: dG_H2O pH:2.92, buffers:formate: 30 mM ; Experimental Assay: dG_H2O pH:9.89, buffers:glycine: 30 mM ; Experimental Assay: dG_H2O buffers:MOPS: 30 mM, pH:6.98 ; Experimental Assay: dG_H2O buffers:formate: 30 mM, pH:3.02 ; Experimental Assay: Cm pH:9.91, buffers:glycine: 30 mM ; Experimental Assay: m pH:9.91, buffers:glycine: 30 mM ; Experimental Assay: dG_H2O pH:9.91, buffers:glycine: 30 mM ; Experimental Assay: Cm pH:2.92, buffers:formate: 30 mM ; Experimental Assay: m pH:2.92, buffers:formate: 30 mM ; Experimental Assay: dG_H2O pH:2.92, buffers:formate: 30 mM ; Experimental Assay: Cm pH:9.89, buffers:glycine: 30 mM ; Experimental Assay: m pH:9.89, buffers:glycine: 30 mM ; Experimental Assay: dG_H2O pH:9.89, buffers:glycine: 30 mM ; Experimental Assay: Cm buffers:MOPS: 30 mM, pH:6.98 ; Experimental Assay: m buffers:MOPS: 30 mM, pH:6.98 ; Experimental Assay: dG_H2O buffers:MOPS: 30 mM, pH:6.98 ; Experimental Assay: Cm buffers:formate: 30 mM, pH:3.02 ; Experimental Assay: m buffers:formate: 30 mM, pH:3.02 ; Experimental Assay: dG_H2O buffers:formate: 30 mM, pH:3.02
Libraries Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV ; 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
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
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

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
100.0 Guanyl-specific ribonuclease T1 P00651 RNT1_ASPOR