Protein stability: functional dependence of denaturational Gibbs energy on urea concentration.


Abstract

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data. Study holds ProTherm entries: 5620, 5621, 5622, 5623, 5624, 5625, 5626, 5627, 5628, 5629, 5630, 5631, 5632, 5633, 5634, 5635, 5636, 5637, 5638, 5639, 5640, 5641, 5642, 5643, 5644 Extra Details: anion binding site

Submission Details

ID: 2eB6ZwEj3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Gupta R;Ahmad F,Biochemistry (1999) Protein stability: functional dependence of denaturational Gibbs energy on urea concentration. PMID:10029541
Additional Information

Study Summary

Number of data points 75
Proteins Ribonuclease pancreatic ; Cytochrome c ; Cytochrome c ; Lysozyme C ; Lysozyme C ; Ribonuclease pancreatic
Unique complexes 3
Assays/Quantities/Protocols Experimental Assay: Cm pH:6.0, ionic:KCl: 0.1 M, buffers:cacodylate: 0.03 M, prot_conc:2mg/ml ; Experimental Assay: m pH:6.0, ionic:KCl: 0.1 M, buffers:cacodylate: 0.03 M, prot_conc:2mg/ml ; Experimental Assay: dG pH:6.0, ionic:KCl: 0.1 M, buffers:cacodylate: 0.03 M, prot_conc:2mg/ml ; Experimental Assay: Cm pH:6.0, buffers:cacodylate: 0.03 M, ionic:KCl: 0.1-1.0 M, prot_conc:2mg/ml ; Experimental Assay: m pH:6.0, buffers:cacodylate: 0.03 M, ionic:KCl: 0.1-1.0 M, prot_conc:2mg/ml ; Experimental Assay: dG pH:6.0, buffers:cacodylate: 0.03 M, ionic:KCl: 0.1-1.0 M, prot_conc:2mg/ml ; Experimental Assay: Cm pH:6.0, buffers:cacodylate: 0.03 M, ionic:KCl: 0.1-1.0 M, prot_conc:2mg/ml ; Experimental Assay: m pH:6.0, buffers:cacodylate: 0.03 M, ionic:KCl: 0.1-1.0 M, prot_conc:2mg/ml ; Experimental Assay: dG pH:6.0, buffers:cacodylate: 0.03 M, ionic:KCl: 0.1-1.0 M, prot_conc:2mg/ml ; Experimental Assay: Cm ionic:KCl: 0.1 M, buffers:glycine-HCl: 0.05 M, pH:3.0, prot_conc:5 mg/ml ; Experimental Assay: m ionic:KCl: 0.1 M, buffers:glycine-HCl: 0.05 M, pH:3.0, prot_conc:5 mg/ml ; Experimental Assay: dG ionic:KCl: 0.1 M, buffers:glycine-HCl: 0.05 M, pH:3.0, prot_conc:5 mg/ml ; Experimental Assay: Cm buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 1.50 M, prot_conc:5 mg/ml ; Experimental Assay: m buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 1.50 M, prot_conc:5 mg/ml ; Experimental Assay: dG buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 1.50 M, prot_conc:5 mg/ml ; Experimental Assay: Cm ionic:KCl: 1.25 M, buffers:glycine-HCl: 0.05 M, pH:3.0, prot_conc:5 mg/ml ; Experimental Assay: m ionic:KCl: 1.25 M, buffers:glycine-HCl: 0.05 M, pH:3.0, prot_conc:5 mg/ml ; Experimental Assay: dG ionic:KCl: 1.25 M, buffers:glycine-HCl: 0.05 M, pH:3.0, prot_conc:5 mg/ml ; Experimental Assay: Cm ionic:KCl: 1.00 M, pH:3.0, prot_conc:5 mg/ml, buffers:glycine-HCl: 0.05 M ; Experimental Assay: m ionic:KCl: 1.00 M, pH:3.0, prot_conc:5 mg/ml, buffers:glycine-HCl: 0.05 M ; Experimental Assay: dG ionic:KCl: 1.00 M, pH:3.0, prot_conc:5 mg/ml, buffers:glycine-HCl: 0.05 M ; Experimental Assay: Cm ionic:KCl: 0.50 M, pH:3.0, prot_conc:5 mg/ml, buffers:glycine-HCl: 0.05 M ; Experimental Assay: m ionic:KCl: 0.50 M, pH:3.0, prot_conc:5 mg/ml, buffers:glycine-HCl: 0.05 M ; Experimental Assay: dG ionic:KCl: 0.50 M, pH:3.0, prot_conc:5 mg/ml, buffers:glycine-HCl: 0.05 M ; Experimental Assay: Cm buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 0.10 M, prot_conc:5 mg/ml ; Experimental Assay: m buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 0.10 M, prot_conc:5 mg/ml ; Experimental Assay: dG buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 0.10 M, prot_conc:5 mg/ml ; Experimental Assay: Cm buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 0.1-1.0 M, prot_conc:5 mg/ml ; Experimental Assay: m buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 0.1-1.0 M, prot_conc:5 mg/ml ; Experimental Assay: dG buffers:glycine-HCl: 0.05 M, pH:3.0, ionic:KCl: 0.1-1.0 M, prot_conc:5 mg/ml ; Experimental Assay: Cm ionic:KCl: 0.75-1.00 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: m ionic:KCl: 0.75-1.00 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: dG ionic:KCl: 0.75-1.00 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: Cm ionic:KCl: 0.30-0.50 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: m ionic:KCl: 0.30-0.50 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: dG ionic:KCl: 0.30-0.50 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: Cm ionic:KCl: 0.1 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: m ionic:KCl: 0.1 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: dG ionic:KCl: 0.1 M, buffers:HCl: , pH:2.0, prot_conc:4 mg/ml ; Experimental Assay: Cm pH:2.0, buffers:HCl: , ionic:KCl: 0.1-1.0 M, prot_conc:4 mg/ml ; Experimental Assay: m pH:2.0, buffers:HCl: , ionic:KCl: 0.1-1.0 M, prot_conc:4 mg/ml ; Experimental Assay: dG pH:2.0, buffers:HCl: , ionic:KCl: 0.1-1.0 M, prot_conc:4 mg/ml
Libraries Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV ; Mutations for sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE ; Mutations for sequence KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL

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
1KTD 2002-01-15T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF CLASS II MHC MOLECULE IEK BOUND TO PIGEON CYTOCHROME C PEPTIDE
2IHL 1993-06-29T00:00:00+0000 1.4 LYSOZYME (E.C.3.2.1.17) (JAPANESE QUAIL)
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
1FBI 1995-01-19T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF A CROSS-REACTION COMPLEX BETWEEN FAB F9.13.7 AND GUINEA-FOWL LYSOZYME
1GHL 1993-05-04T00:00:00+0000 2.1 THE THREE-DIMENSIONAL STRUCTURE OF PHEASANT AND GUINEA-FOWL EGG LYSOZYMES
1HHL 1993-05-04T00:00:00+0000 1.9 THE THREE-DIMENSIONAL STRUCTURE OF PHEASANT AND GUINEA-FOWL EGG LYSOZYMES
1JHL 1993-05-04T00:00:00+0000 2.4 THREE-DIMENSIONAL STRUCTURE OF A HETEROCLITIC ANTIGEN-ANTIBODY CROSS-REACTION COMPLEX
1BQL 1995-02-03T00:00:00+0000 2.6 STRUCTURE OF AN ANTI-HEL FAB FRAGMENT COMPLEXED WITH BOBWHITE QUAIL LYSOZYME
1DKJ 1996-01-10T00:00:00+0000 2.0 BOBWHITE QUAIL LYSOZYME
1DKK 1996-01-10T00:00:00+0000 1.9 BOBWHITE QUAIL LYSOZYME WITH NITRATE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
90.3 Cytochrome c B4USV4 CYC_OTOGA
91.3 Cytochrome c P00021 CYC_COLLI
92.2 Cytochrome c P00020 CYC_ANAPL
92.3 Cytochrome c P81280 CYC_ALLMI
90.5 Cytochrome c Q52V10 CYC_SAISC
93.3 Cytochrome c P00012 CYC_MIRLE
93.3 Cytochrome c Q52V09 CYC_CEPBA
93.3 Cytochrome c P00013 CYC_MINSC
93.3 Cytochrome c P00014 CYC_MACGI
94.3 Cytochrome c P00011 CYC_CANLF
94.3 Cytochrome c P62898 CYC_RAT
94.3 Cytochrome c P00008 CYC_RABIT
94.3 Cytochrome c P62897 CYC_MOUSE
95.2 Cytochrome c P68098 CYC_LAMGU
95.2 Cytochrome c P68100 CYC_ESCRO
95.2 Cytochrome c P68099 CYC_CAMDR
94.3 Cytochrome c P00007 CYC_HIPAM
97.1 Cytochrome c P62896 CYC_SHEEP
97.1 Cytochrome c P62895 CYC_PIG
97.1 Cytochrome c P62894 CYC_BOVIN
99.0 Cytochrome c P68096 CYC_EQUBU
99.0 Cytochrome c P68097 CYC_EQUAS
100.0 Cytochrome c P00004 CYC_HORSE
93.0 Lysozyme C P81711 LYSC_SYRSO
92.3 Lysozyme C P49663 LYSC_PHAVE
94.6 Lysozyme C P24533 LYSC_SYRRE
93.0 Lysozyme C P24364 LYSC_LOPLE
92.2 Lysozyme C P00704 LYSC_NUMME
95.3 Lysozyme C Q7LZT2 LYSC_TRATE
95.3 Lysozyme C P22910 LYSC_CHRAM
96.1 Lysozyme C P19849 LYSC_PAVCR
96.1 Lysozyme C Q7LZI3 LYSC_TRASA
96.9 Lysozyme C Q7LZP9 LYSC_LOPIM
96.9 Lysozyme C Q7LZQ0 LYSC_CATWA
96.9 Lysozyme C P00699 LYSC_CALCC
96.9 Lysozyme C P00700 LYSC_COLVI
93.2 Lysozyme C P00702 LYSC_PHACO
95.2 Lysozyme C P00703 LYSC_MELGA
95.3 Lysozyme C P00701 LYSC_COTJA
100.0 Lysozyme C P00698 LYSC_CHICK