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Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule.
Biochemistry. 1994 Jan 11; 33(1):106-15.B

Abstract

The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.

Authors+Show Affiliations

Khurana R
National Centre for Biological Sciences, Indian Institute of Science Campus, Bangalore.
Udgaonkar JB
No affiliation info available

MeSH

BacillusBacterial ProteinsChromatography, GelChromatography, Ion ExchangeCircular DichroismElectrophoresis, Polyacrylamide GelEscherichia coliGenes, BacterialHydrogen-Ion ConcentrationKineticsProtein ConformationProtein DenaturationProtein FoldingRecombinant ProteinsRibonucleasesSpectrometry, FluorescenceSpectrophotometry, UltravioletThermodynamicsUrea

Pub Type(s)

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

Language

eng

PubMed ID

8286327

Citation

Khurana, R, and J B. Udgaonkar. "Equilibrium Unfolding Studies of Barstar: Evidence for an Alternative Conformation Which Resembles a Molten Globule." Biochemistry, vol. 33, no. 1, 1994, pp. 106-15.
Khurana R, Udgaonkar JB. Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule. Biochemistry. 1994;33(1):106-15.
Khurana, R., & Udgaonkar, J. B. (1994). Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule. Biochemistry, 33(1), 106-15.
Khurana R, Udgaonkar JB. Equilibrium Unfolding Studies of Barstar: Evidence for an Alternative Conformation Which Resembles a Molten Globule. Biochemistry. 1994 Jan 11;33(1):106-15. PubMed PMID: 8286327.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule. AU - Khurana,R, AU - Udgaonkar,J B, PY - 1994/1/11/pubmed PY - 1994/1/11/medline PY - 1994/1/11/entrez SP - 106 EP - 15 JF - Biochemistry JO - Biochemistry VL - 33 IS - 1 N2 - The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8286327/Equilibrium_unfolding_studies_of_barstar:_evidence_for_an_alternative_conformation_which_resembles_a_molten_globule_ DB - PRIME DP - Unbound Medicine ER -