title: 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.
authors.affiliation: Department of Medical Biochemistry and Genetics, Texas A&M University, College Station 77843-1114.
authors.forename: C N
authors.initials: CN
authors.lastname: Pace
authors.forename: D V
authors.initials: DV
authors.lastname: Laurents
authors.forename: R E
authors.initials: RE
authors.lastname: Erickson
mesh_terms: D001426:Bacterial Proteins; D004220:Disulfides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D006163:Ribonuclease T1 / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014508:Urea / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D014443:Tyrosine; D014364:Tryptophan; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D006163:Ribonuclease T1