Thermodynamics of denaturant-induced unfolding of a protein that exhibits variable two-state denaturation.


Abstract

Free energy changes (DeltaG(degrees)(N-->D)) obtained by denaturant-induced unfolding using the linear extrapolation method (LEM) are presumed to reflect the stability differences between native (N) and denatured (D) species in the absence of denaturant. It has been shown that with urea and guanidine hydrochloride (GdnHCl) some proteins exhibit denaturant-independent (DeltaG(degrees)(N-->D)). But with several other proteins urea and GdnHCl give different (DeltaG(degrees)(N-->D)) values for the same protein, meaning that the free energy difference between N and D is not the only contribution to one or both (DeltaG(degrees)(N-->D)) values. Using beta1, a mutant form of the protein G B1 domain, we show that both urea- and GdnHCl-induced denaturations are two-state and reversible but that the denaturants give different values for (DeltaG(degrees)(N-->D)). While spectral observables are sensitive to the shift between N and D states (between states effect), they are not sensitive to denaturant-induced changes that occur within the individual N and D states (within state effect). By contrast, nonspectral observables such as Stokes radius and thermodynamic observables such as proton uptake/release are often sensitive to both "between states" and "within state" effects. These observables, along with spectral measurements, provide descriptions of urea- and GdnHCl-induced denaturation of beta1. Our results suggest that in the predenaturation concentration range GdnHCl changes the free energy of the native ensemble in a nonlinear manner but that urea does not. As with RNase A and beta-lactoglobulin, beta1 exhibits variable two-state behavior with GdnHCl-induced denaturation in that the free energy of the native ensemble in the predenaturation zone changes (varies) with GdnHCl concentration in a nonlinear manner.

Submission Details

ID: BNg4M8Mw3

Submitter: Connie Wang

Submission Date: Aug. 1, 2017, 1:27 p.m.

Version: 1

Publication Details
Ferreon AC;Bolen DW,Biochemistry (2004) Thermodynamics of denaturant-induced unfolding of a protein that exhibits variable two-state denaturation. PMID:15491142
Additional Information

Study Summary

Number of data points 78
Proteins Immunoglobulin G-binding protein G
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: Cm-GdnHCL Denaturation-Fluorescence(275,340) ; Experimental Assay: Cm-Urea Denaturation-tyrosine exposure ; Experimental Assay: ∆G-GdnHCL Denaturation-Fluorescence(295,340) ; Experimental Assay: ∆G-GdnHCL Denaturation-far UV CD (222nm)-Operator ; Experimental Assay: Cm-Urea Denaturation-Fluorescence(295,340) ; Experimental Assay: ∆G-Urea Denaturation-tyrosine exposure ; Experimental Assay: ∆G-Urea Denaturation-1/Kd (size exclusion) ; Experimental Assay: m-value-Urea Denaturation-Fluorescence(275,340) ; Experimental Assay: ∆G-Urea Denaturation-Fluorescence(275,340) ; Experimental Assay: ∆G-Urea Denaturation-Fluorescence(295,340) ; Experimental Assay: ∆G-Urea Denaturation-far UV CD (222nm) ; Experimental Assay: m-value-Urea Denaturation-Fluorescence(295,340) ; Experimental Assay: m-value-Urea Denaturation-far UV CD (222nm) ; Experimental Assay: m-value-Urea Denaturation-second-derivative UV ; Experimental Assay: m-value-Urea Denaturation-tyrosine exposure ; Experimental Assay: m-value-Urea Denaturation-1/Kd (size exclusion) ; Experimental Assay: m-value-Urea Denaturation-Stokes Radius ; Experimental Assay: m-value-GdnHCL Denaturation-second derivative UV ; Experimental Assay: m-value-GdnHCL Denaturation-Fluorescence(295,340) ; Experimental Assay: ∆G-Urea Denaturation-Stokes Radius ; Experimental Assay: Cm-GdnHCL Denaturation-Fluorescence(295,340) ; Experimental Assay: Cm-Urea Denaturation-second-derivative UV ; Experimental Assay: ∆G-GdnHCL Denaturation-Fluorescence(275,340) ; Experimental Assay: Cm-Urea Denaturation-Stokes Radius ; Experimental Assay: Cm-GdnHCL Denaturation-second derivative UV ; Experimental Assay: Cm-Urea Denaturation-far UV CD (222nm) ; Experimental Assay: Cm-GdnHCL Denaturation-far UV CD (222nm)-NLSQ ; Experimental Assay: m-value-GdnHCL Denaturation-tyrosine exposure ; Experimental Assay: m-value-GdnHCL Denaturation-far UV CD (222nm)-NLSQ ; Experimental Assay: ∆G-GdnHCL Denaturation-far UV CD (222nm)-NLSQ ; Experimental Assay: ∆G-Urea Denaturation-second-derivative UV ; Experimental Assay: ∆G-GdnHCL Denaturation-second derivative UV ; Experimental Assay: m-value-GdnHCL Denaturation-far UV CD (222nm)-Operator ; Experimental Assay: m-value-GdnHCL Denaturation-Fluorescence(275,340) ; Experimental Assay: Cm-GdnHCL Denaturation-far UV CD (222nm)-Operator ; Experimental Assay: Cm-GdnHCL Denaturation-tyrosine exposure ; Experimental Assay: Cm-Urea Denaturation-1/Kd (size exclusion) ; Experimental Assay: ∆G-GdnHCL Denaturation-tyrosine exposure ; Experimental Assay: Cm-Urea Denaturation-Fluorescence(275,340)

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1MPE 2002-10-30 Ensemble of 20 structures of the tetrameric mutant of the B1 domain of streptococcal protein G
2MBB 2014-06-04 Solution Structure of the human Polymerase iota UBM1-Ubiquitin Complex
1LE3 2002-04-24 NMR Structure of Tryptophan Zipper 4: A Stable Beta-Hairpin Peptide Based on the C-terminal Hairpin of the B1 Domain of Protein G
2LGI 2011-10-26 Atomic Resolution Protein Structures using NMR Chemical Shift Tensors
2J53 2007-09-25 Solution Structure of GB1 domain Protein G and low and high pressure.
2PLP 2007-05-08 Ultra high resolution backbone conformation of protein GB1 from residual dipolar couplings alone
2GB1 1993-04-15 A NOVEL, HIGHLY STABLE FOLD OF THE IMMUNOGLOBULIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G
2NMQ 2006-11-21 Simultaneous determination of protein structure and dynamics using rdcs
3GB1 1999-06-23 STRUCTURES OF B1 DOMAIN OF STREPTOCOCCAL PROTEIN G
2J52 2007-09-25 Solution Structure of GB1 domain Protein G and low and high pressure.
1Q10 2003-10-14 Ensemble of 40 Structures of the Dimeric Mutant of the B1 Domain of Streptococcal Protein G
1GB1 1993-04-15 A NOVEL, HIGHLY STABLE FOLD OF THE IMMUNOGLOBULIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G
2N7J 2015-10-14 Sidechain chi1 distribution in B3 domain of protein G from extensive sets of residual dipolar couplings
2RPV 2009-09-15 Solution Structure of GB1 with LBT probe
1PN5 2003-10-07 NMR structure of the NALP1 Pyrin domain (PYD)
2IGH 1994-01-31 DETERMINATION OF THE SOLUTION STRUCTURES OF DOMAINS II AND III OF PROTEIN G FROM STREPTOCOCCUS BY 1H NMR
2RMM 2007-12-04 Solution structure of GB1 A34F mutant
2K0P 2009-03-03 Determination of a Protein Structure in the Solid State from NMR Chemical Shifts
2KLK 2009-10-06 Solution structure of GB1 A34F mutant with RDC and SAXS
2KBT 2009-02-03 Attachment of an NMR-invisible solubility enhancement tag (INSET) using a sortase-mediated protein ligation method
2IGD 1998-07-29 1.1 ANISOTROPIC STRUCTURE OF PROTEIN G IGG-BINDING DOMAIN III AT 1.1 ANGSTROM RESOLUTION
1IGD 1994-11-01 1.1 THE THIRD IGG-BINDING DOMAIN FROM STREPTOCOCCAL PROTEIN G: AN ANALYSIS BY X-RAY CRYSTALLOGRAPHY OF THE STRUCTURE ALONE AND IN A COMPLEX WITH FAB
6CNE 2019-07-10 1.2 Selenomethionine variant (V29SeM) of protein GB1
3MP9 2011-02-23 1.2 Structure of Streptococcal protein G B1 domain at pH 3.0
1PGX 1992-07-15 1.66 THE 1.66 ANGSTROMS X-RAY STRUCTURE OF THE B2 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G AND COMPARISON TO THE NMR STRUCTURE OF THE B1 DOMAIN
6L9D 2020-08-12 1.73 X-ray structure of synthetic GB1 domain with mutations K10(DVA), T11S
6LJI 2020-08-12 1.84 X-ray structure of synthetic GB1 domain with mutations K10(DVA), T11V
6L91 2020-08-12 1.84 X-ray structure of synthetic GB1 domain with the mutation K10(DVA)
1PGB 1994-04-30 1.92 TWO CRYSTAL STRUCTURES OF THE B1 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCCOCAL PROTEIN G AND COMPARISON WITH NMR
6L9B 2020-08-12 1.95 X-ray structure of synthetic GB1 domain with mutations K10(DVA), T11A
4KGS 2013-09-04 1.95 Backbone Modifications in the Protein GB1 Loops: beta-3-Val21, beta-3-Asp40
1EM7 2002-05-08 2.0 HELIX VARIANT OF THE B1 DOMAIN FROM STREPTOCOCCAL PROTEIN G
4KGR 2013-09-04 2.0 Backbone Modifications in the Protein GB1 Helix: beta-3-Ala24, beta-3-Lys28, beta-3-Lys31, beta-3-Asn35
4KGT 2013-09-04 2.0 Backbone Modifications in the Protein GB1 Turns: Aib10, D-Pro47
1PGA 1994-04-30 2.07 TWO CRYSTAL STRUCTURES OF THE B1 IMMUNOGLOBULIN-BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G AND COMPARISON WITH NMR
1MVK 2002-10-30 2.5 X-ray structure of the tetrameric mutant of the B1 domain of streptococcal protein G
1IGC 1995-06-03 2.6 IGG1 FAB FRAGMENT (MOPC21) COMPLEX WITH DOMAIN III OF PROTEIN G FROM STREPTOCOCCUS
6V9I 2020-04-29 5.2 cryo-EM structure of Cullin5 bound to RING-box protein 2 (Cul5-Rbx2)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Immunoglobulin G-binding protein G P06654 SPG1_STRSG