Thermodynamics of denaturation of hisactophilin, a beta-trefoil protein.


Abstract

Hisactophilin is a histidine-rich pH-dependent actin-binding protein from Dictyostelium discoideum. The structure of hisactophilin is typical of the beta-trefoil fold, a common structure adopted by diverse proteins with unrelated primary sequences and functions. The thermodynamics of denaturation of hisactophilin have been measured using fluorescence- and CD-monitored equilibrium urea denaturation curves, pH-denaturation, and thermal denaturation curves, as well as differential scanning calorimetry. Urea denaturation is reversible from pH 5.7 to pH 9.7; however, thermal denaturation is highly reversible only below pH approximately 6.2. Reversible denaturation by urea and heat is well fit using a two-state transition between the native and the denatured states. Urea denaturation curves are best fit using a quadratic dependence of the Gibbs free energy of unfolding upon urea concentration. Hisactophilin has moderate, roughly constant stability from pH 7.7 to pH 9.7; however, below pH 7.7, stability decreases markedly, most likely due to protonation of histidine residues. Enthalpic effects of histidine ionization upon unfolding also appear to be involved in the occurrence of cold unfolding of hisactophilin under relatively mild solution conditions. The stability data for hisactophilin are compared with data on hisactophilin function, and with data for two other beta-trefoil proteins, human interleukin-1beta, and basic fibroblast growth factor. Study holds ProTherm entries: 11101, 11102, 11103, 11104, 11105, 11106, 11107, 11108, 11109, 11110, 11111, 11112, 11113, 11114, 11115, 11116, 11117, 11118, 11119, 11120, 11121, 11122, 11123, 11124, 11125, 11126, 11127, 11128, 11129, 11130 Extra Details: additive : EDTA(1 mM),There are thermodynamic data too using binomial extrapolation method histidine-rich pH-dependent actin-binding protein;,beta-trefoil fold; two-state transition

Submission Details

ID: xcen3Y4m3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Liu C;Chu D;Wideman RD;Houliston RS;Wong HJ;Meiering EM,Biochemistry (2001) Thermodynamics of denaturation of hisactophilin, a beta-trefoil protein. PMID:11300762
Additional Information

Number of data points 72
Proteins Hisactophilin-1 ; Hisactophilin-1
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp pH:6.25 ; Experimental Assay: dG pH:6.25 ; Experimental Assay: dCp pH:6.09 ; Experimental Assay: dG pH:6.09 ; Experimental Assay: dCp pH:6.03 ; Experimental Assay: dG pH:6.03 ; Experimental Assay: dCp pH:5.81 ; Experimental Assay: dG pH:5.81 ; Experimental Assay: dCp pH:5.75 ; Experimental Assay: dG pH:5.75 ; Experimental Assay: dCp pH:5.59 ; Experimental Assay: dG pH:5.59 ; Experimental Assay: Tm pH:6.25 ; Experimental Assay: Tm pH:6.09 ; Experimental Assay: Tm pH:6.03 ; Experimental Assay: Tm pH:5.81 ; Experimental Assay: Tm pH:5.75 ; Experimental Assay: Tm pH:5.59 ; Experimental Assay: Cm pH:9.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: m pH:9.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: dG_H2O pH:9.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: Cm pH:9.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: m pH:9.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: dG_H2O pH:9.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: Cm ionic:NaCl: 400 mM, buffers:Glycine/sodium glycine: 50 mM, pH:8.7 ; Experimental Assay: m ionic:NaCl: 400 mM, buffers:Glycine/sodium glycine: 50 mM, pH:8.7 ; Experimental Assay: dG_H2O ionic:NaCl: 400 mM, buffers:Glycine/sodium glycine: 50 mM, pH:8.7 ; Experimental Assay: Cm buffers:Glycine/sodium glycine: 50 mM, pH:8.7 ; Experimental Assay: m buffers:Glycine/sodium glycine: 50 mM, pH:8.7 ; Experimental Assay: dG_H2O buffers:Glycine/sodium glycine: 50 mM, pH:8.7 ; Experimental Assay: Cm pH:8.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: m pH:8.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: dG_H2O pH:8.7, buffers:Glycine/sodium glycine: 50 mM ; Experimental Assay: Cm pH:8.15, buffers:KH2PO4/K2HPO4: 50 mM ; Experimental Assay: m pH:8.15, buffers:KH2PO4/K2HPO4: 50 mM ; Experimental Assay: dG_H2O pH:8.15, buffers:KH2PO4/K2HPO4: 50 mM ; Experimental Assay: Cm buffers:KH2PO4/K2HPO4: 50 mM, pH:7.7 ; Experimental Assay: m buffers:KH2PO4/K2HPO4: 50 mM, pH:7.7 ; Experimental Assay: dG_H2O buffers:KH2PO4/K2HPO4: 50 mM, pH:7.7 ; Experimental Assay: Cm buffers:KH2PO4/K2HPO4: 50 mM, pH:7.7 ; Experimental Assay: m buffers:KH2PO4/K2HPO4: 50 mM, pH:7.7 ; Experimental Assay: dG_H2O buffers:KH2PO4/K2HPO4: 50 mM, pH:7.7 ; Experimental Assay: Cm pH:7.08, buffers:KH2PO4/K2HPO4: 50 mM, temp:5.0 C ; Experimental Assay: m pH:7.08, buffers:KH2PO4/K2HPO4: 50 mM, temp:5.0 C ; Experimental Assay: dG_H2O pH:7.08, buffers:KH2PO4/K2HPO4: 50 mM, temp:5.0 C ; Experimental Assay: Cm buffers:KH2PO4/K2HPO4: 50 mM, pH:6.7 ; Experimental Assay: m buffers:KH2PO4/K2HPO4: 50 mM, pH:6.7 ; Experimental Assay: dG_H2O buffers:KH2PO4/K2HPO4: 50 mM, pH:6.7 ; Experimental Assay: Cm buffers:KH2PO4/K2HPO4: 50 mM, pH:6.7 ; Experimental Assay: m buffers:KH2PO4/K2HPO4: 50 mM, pH:6.7 ; Experimental Assay: dG_H2O buffers:KH2PO4/K2HPO4: 50 mM, pH:6.7 ; Experimental Assay: Cm pH:6.2, buffers:MES: 50 mM, ionic:NaCl: 400 mM ; Experimental Assay: m pH:6.2, buffers:MES: 50 mM, ionic:NaCl: 400 mM ; Experimental Assay: dG_H2O pH:6.2, buffers:MES: 50 mM, ionic:NaCl: 400 mM ; Experimental Assay: Cm pH:6.2, buffers:MES: 50 mM ; Experimental Assay: m pH:6.2, buffers:MES: 50 mM ; Experimental Assay: dG_H2O pH:6.2, buffers:MES: 50 mM ; Experimental Assay: Cm buffers:MES: 50 mM, temp:5.0 C, pH:5.87 ; Experimental Assay: m buffers:MES: 50 mM, temp:5.0 C, pH:5.87 ; Experimental Assay: dG_H2O buffers:MES: 50 mM, temp:5.0 C, pH:5.87 ; Experimental Assay: Cm pH:5.7, buffers:MES: 50 mM ; Experimental Assay: m pH:5.7, buffers:MES: 50 mM ; Experimental Assay: dG_H2O pH:5.7, buffers:MES: 50 mM ; Experimental Assay: Cm pH:5.7, buffers:KH2PO4/K2HPO4: 50 mM ; Experimental Assay: m pH:5.7, buffers:KH2PO4/K2HPO4: 50 mM ; Experimental Assay: dG_H2O pH:5.7, buffers:KH2PO4/K2HPO4: 50 mM ; Experimental Assay: Cm pH:5.7, buffers:MES: 50 mM ; Experimental Assay: m pH:5.7, buffers:MES: 50 mM ; Experimental Assay: dG_H2O pH:5.7, buffers:MES: 50 mM ; Experimental Assay: Cm pH:5.7, buffers:KH2PO4/K2HPO4: 50 mM ; Experimental Assay: m pH:5.7, buffers:KH2PO4/K2HPO4: 50 mM ; Experimental Assay: dG_H2O pH:5.7, buffers:KH2PO4/K2HPO4: 50 mM
Libraries Mutations for sequence MGNRAFKSHHGHFLSAEGEAVKTHHGHHDHHTHFHVENHGGKVALKTHCGKYLSIGDHKQVYLSHHLHGDHSLFHLEHHGGKVSIKGHHHHYISADHHGHVSTKEHHDHDTTFEEIII
Sequence Assay Result Units