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. Study holds ProTherm entries: 18232, 18233, 18234, 18235, 18236, 18237, 18238 Extra Details: Fluorescen T 2 Q, I 6 A, T 44 Ace lambda(ex) = 275 nm and lambda(em) = 340 nm free energy change; Stokes radius; non-linear

Submission Details

ID: EZNwsw6e

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 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 21
Proteins Immunoglobulin G-binding protein G ; Immunoglobulin G-binding protein G
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm prot_conc:0.2-0.3 mg/ml ; Experimental Assay: m prot_conc:0.2-0.3 mg/ml ; Experimental Assay: dG_H2O prot_conc:0.2-0.3 mg/ml ; Experimental Assay: Cm prot_conc:0.2-0.3 mg/ml ; Experimental Assay: m prot_conc:0.2-0.3 mg/ml ; Experimental Assay: dG_H2O prot_conc:0.2-0.3 mg/ml ; Experimental Assay: Cm prot_conc:5-10 micro g/ml ; Experimental Assay: m prot_conc:5-10 micro g/ml ; Experimental Assay: dG_H2O prot_conc:5-10 micro g/ml ; Experimental Assay: Cm prot_conc:5-10 micro g/ml ; Experimental Assay: m prot_conc:5-10 micro g/ml ; Experimental Assay: dG_H2O prot_conc:5-10 micro g/ml
Libraries Mutations for sequence MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE

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 UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Immunoglobulin G-binding protein G P06654 SPG1_STRSG
100.0 Immunoglobulin G-binding protein G P19909 SPG2_STRSG