Urea denaturation of barnase: pH dependence and characterization of the unfolded state.


Abstract

To investigate the pH dependence of the conformational stability of barnase, urea denaturation curves were determined over the pH range 2-10. The maximum conformational stability of barnase is 9 kcal mol-1 and occurs between pH 5 and 6. The dependence of delta G on urea concentration increases from 1850 cal mol-1 M-1 at high pH to about 3000 cal mol-1 M-1 near pH 3. This suggests that the unfolded conformations of barnase become more accessible to urea as the net charge on the molecule increases. Previous studies suggested that in 8 M urea barnase unfolds more completely than ribonuclease T1, even with the disulfide bonds broken [Pace, C.N., Laurents, D. V., & Thomson, J.A. (1990) Biochemistry 29, 2564-2572]. In support of this, solvent perturbation difference spectroscopy showed that in 8 M urea the Trp and Tyr residues in barnase are more accessible to perturbation by dimethyl sulfoxide than in ribonuclease T1 with the disulfide bonds broken. Study holds ProTherm entries: 4015, 4016, 4017, 4018, 4019, 4020, 4021 Extra Details: pH dependence; accessible; disulfide bonds;,solvent perturbation

Submission Details

ID: 9uMrzDGS

Submitter: Connie Wang

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

Version: 1

Publication Details
Pace CN;Laurents DV;Erickson RE,Biochemistry (1992) Urea denaturation of barnase: pH dependence and characterization of the unfolded state. PMID:1547213
Additional Information

Number of data points 21
Proteins Ribonuclease ; Ribonuclease
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm temp:20.0 C, pH:2.5, buffers:diglycine: 100 mM ; Experimental Assay: m temp:20.0 C, pH:2.5, buffers:diglycine: 100 mM ; Experimental Assay: dG_H2O temp:20.0 C, pH:2.5, buffers:diglycine: 100 mM ; Experimental Assay: Cm pH:2.98, temp:20.0 C, buffers:diglycine: 100 mM ; Experimental Assay: m pH:2.98, temp:20.0 C, buffers:diglycine: 100 mM ; Experimental Assay: dG_H2O pH:2.98, temp:20.0 C, buffers:diglycine: 100 mM ; Experimental Assay: Cm pH:2.76, buffers:diglycine: 30 mM, temp:20.0 C, ionic:NaCl: 0.5 M ; Experimental Assay: m pH:2.76, temp:20.0 C, buffers:diglycine: 30 mM, ionic:NaCl: 0.5 M ; Experimental Assay: dG_H2O pH:2.76, buffers:diglycine: 30 mM, temp:20.0 C, ionic:NaCl: 0.5 M ; Experimental Assay: Cm buffers:diglycine: 30 mM, ionic:NaCl: 0.1 M, pH:2.82, temp:20.0 C ; Experimental Assay: m temp:20.0 C, ionic:NaCl: 0.1 M, pH:2.82, buffers:diglycine: 30 mM ; Experimental Assay: dG_H2O buffers:diglycine: 30 mM, ionic:NaCl: 0.1 M, pH:2.82, temp:20.0 C ; Experimental Assay: Cm buffers:diglycine: 30 mM, pH:2.87, temp:20.0 C ; Experimental Assay: m temp:20.0 C, pH:2.87, buffers:diglycine: 30 mM ; Experimental Assay: dG_H2O buffers:diglycine: 30 mM, pH:2.87, temp:20.0 C ; Experimental Assay: Cm pH:3.17, buffers:diglycine: 30 mM, temp:20.0 C ; Experimental Assay: m pH:3.17, temp:20.0 C, buffers:diglycine: 30 mM ; Experimental Assay: dG_H2O pH:3.17, buffers:diglycine: 30 mM, temp:20.0 C ; Experimental Assay: Cm buffers:MOPS: 30 mM, temp:25.0 C, pH:7.0 ; Experimental Assay: m buffers:MOPS: 30 mM, temp:25.0 C, pH:7.0 ; Experimental Assay: dG_H2O buffers:MOPS: 30 mM, temp:25.0 C, pH:7.0
Libraries Mutations for sequence AQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR
Sequence Assay Result Units