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Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation.
Biochim Biophys Acta. 2003 May 30; 1648(1-2):184-94.BB

Abstract

The conformational stability of RNase Rs was determined with chemical and thermal denaturants over the pH range of 1-10. Equilibrium unfolding with urea showed that values of D(1/2) (5.7 M) and DeltaG(H(2)O) (12.8 kcal/mol) were highest at pH 5.0, its pI and the maximum conformational stability of RNase Rs was observed near pH 5.0. Denaturation with guanidine hydrochloride (GdnHCl), at pH 5.0, gave similar values of DeltaG(H(2)O) although GdnHCl was 2-fold more potent denaturant with D(1/2) value of 3.1 M. The curves of fraction unfolded (f(U)) obtained with fluorescence and CD measurements overlapped at pH 5.0. Denaturation of RNase Rs with urea in the pH range studied was reversible but the enzyme denatured irreversibly >pH 11.0. Thermal denaturation of RNase Rs was reversible in the pH range of 2.0-3.0 and 6.0-9.0. Thermal denaturation in the pH range 4.0-5.5 resulted in aggregation and precipitation of the protein above 55 degrees C. The aggregate was amorphous or disordered precipitate as observed in TE micrographs. Blue shift in emission lambda(max) and enhancement of fluorescence intensity of ANS at 70 degrees C indicated the presence of solvent exposed hydrophobic surfaces as a result of heat treatment. Aggregation could be prevented partially with alpha-cyclodextrin (0.15 M) and completely with urea at concentrations >3 M. Aggregation was probably due to intermolecular hydrophobic interaction favored by minimum charge-charge repulsion at the pI of the enzyme. Both urea and temperature-induced denaturation studies showed that RNase Rs unfolds through a two-state F right arrow over left arrow U mechanism. The pH dependence of stability described by DeltaG(H(2)O) (urea) and DeltaG (25 degrees C) suggested that electrostatic interactions among the charged groups make a significant contribution to the conformational stability of RNase Rs. Since RNase Rs is a disulfide-containing protein, the major element for structural stability are the covalent disulfide bonds.

Authors+Show Affiliations

Deshpande RA
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India.
Khan MI
No affiliation info available
Shankar V
No affiliation info available

MeSH

Circular DichroismEnzyme StabilityGuanidineHot TemperatureHydrogen-Ion ConcentrationProtein DenaturationProtein FoldingRhizopusRibonucleasesSpectrometry, FluorescenceUrea

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12758161

Citation

Deshpande, Rajashree A., et al. "Equilibrium Unfolding of RNase Rs From Rhizopus Stolonifer: pH Dependence of Chemical and Thermal Denaturation." Biochimica Et Biophysica Acta, vol. 1648, no. 1-2, 2003, pp. 184-94.
Deshpande RA, Khan MI, Shankar V. Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation. Biochim Biophys Acta. 2003;1648(1-2):184-94.
Deshpande, R. A., Khan, M. I., & Shankar, V. (2003). Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation. Biochimica Et Biophysica Acta, 1648(1-2), 184-94.
Deshpande RA, Khan MI, Shankar V. Equilibrium Unfolding of RNase Rs From Rhizopus Stolonifer: pH Dependence of Chemical and Thermal Denaturation. Biochim Biophys Acta. 2003 May 30;1648(1-2):184-94. PubMed PMID: 12758161.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation. AU - Deshpande,Rajashree A, AU - Khan,M Islam, AU - Shankar,Vepatu, PY - 2003/5/22/pubmed PY - 2003/7/4/medline PY - 2003/5/22/entrez SP - 184 EP - 94 JF - Biochimica et biophysica acta JO - Biochim Biophys Acta VL - 1648 IS - 1-2 N2 - The conformational stability of RNase Rs was determined with chemical and thermal denaturants over the pH range of 1-10. Equilibrium unfolding with urea showed that values of D(1/2) (5.7 M) and DeltaG(H(2)O) (12.8 kcal/mol) were highest at pH 5.0, its pI and the maximum conformational stability of RNase Rs was observed near pH 5.0. Denaturation with guanidine hydrochloride (GdnHCl), at pH 5.0, gave similar values of DeltaG(H(2)O) although GdnHCl was 2-fold more potent denaturant with D(1/2) value of 3.1 M. The curves of fraction unfolded (f(U)) obtained with fluorescence and CD measurements overlapped at pH 5.0. Denaturation of RNase Rs with urea in the pH range studied was reversible but the enzyme denatured irreversibly >pH 11.0. Thermal denaturation of RNase Rs was reversible in the pH range of 2.0-3.0 and 6.0-9.0. Thermal denaturation in the pH range 4.0-5.5 resulted in aggregation and precipitation of the protein above 55 degrees C. The aggregate was amorphous or disordered precipitate as observed in TE micrographs. Blue shift in emission lambda(max) and enhancement of fluorescence intensity of ANS at 70 degrees C indicated the presence of solvent exposed hydrophobic surfaces as a result of heat treatment. Aggregation could be prevented partially with alpha-cyclodextrin (0.15 M) and completely with urea at concentrations >3 M. Aggregation was probably due to intermolecular hydrophobic interaction favored by minimum charge-charge repulsion at the pI of the enzyme. Both urea and temperature-induced denaturation studies showed that RNase Rs unfolds through a two-state F right arrow over left arrow U mechanism. The pH dependence of stability described by DeltaG(H(2)O) (urea) and DeltaG (25 degrees C) suggested that electrostatic interactions among the charged groups make a significant contribution to the conformational stability of RNase Rs. Since RNase Rs is a disulfide-containing protein, the major element for structural stability are the covalent disulfide bonds. SN - 0006-3002 UR - https://www.unboundmedicine.com/medline/citation/12758161/Equilibrium_unfolding_of_RNase_Rs_from_Rhizopus_stolonifer:_pH_dependence_of_chemical_and_thermal_denaturation_ DB - PRIME DP - Unbound Medicine ER -