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

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
Sequence Assay Result Units