Thermodynamic analysis of the chemotactic protein from Escherichia coli, CheY.

Abstract

CheY, the 129 amino acid chemotactic protein from Escherichia coli, is a good model for studies of folding of parallel alpha/beta proteins. We report here the thermodynamic characterization of the wild-type CheY at different pH values and in different buffers and denaturation conditions. The denaturation of CheY by urea monitored by circular dichroism and fluorescence fits the two-state unfolding model. The stability of the protein is ionic strength dependent, probably due to the presence of three Asp residues in very close proximity in its active site. The presence of a Mg2+ ion, which seems to interact with Asp 13 in the active site, stabilizes the native structure by up to 6.9 kJ mol-1. The CheY maximum stability (31.7 +/- 2.1 kJ mol-1), without magnesium, is reached at pH 5.1. Analysis of scanning calorimetry data has shown that temperature-induced unfolding of CheY is not a two-state process and proceeds through a highly populated intermediate state, corresponding to protein dimers, as was subsequently confirmed by direct cross-linking experiments. According to circular dichroism, fluorescence, nuclear magnetic resonance, and ANS binding experiments, this "intermediate dimer" at pH 2.5 exhibits all known characteristics of the "molten globule" state. The reversible dimerization of "molten globules" might explain such peculiarities as the increased stability or the cooperative unfolding found for the molten globule state of some proteins. Study holds ProTherm entries: 2809, 2810, 2811, 11821, 11822 Extra Details: additive : EDTA(1 mM), CheY; chemotactic protein; alpha/beta proteins; molten globule;,temperature-induced unfolding

Submission Details

ID: UyP2m7n64

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:20 p.m.

Version: 1

Publication Details
Filimonov VV;Prieto J;Martinez JC;Bruix M;Mateo PL;Serrano L,Biochemistry (1993) Thermodynamic analysis of the chemotactic protein from Escherichia coli, CheY. PMID:8251514
Additional Information

Study Summary

Number of data points 13
Proteins Chemotaxis protein CheY ; Chemotaxis protein CheY
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp buffers:glycine or CAPS: 5 mM or 10 mM, temp:25.0 C, pH:10.3 ; Experimental Assay: dG buffers:glycine or CAPS: 5 mM or 10 mM, pH:10.3 ; Experimental Assay: dCp buffers:glycine or acetate: 5 mM or 10 mM, pH:2.5, temp:25.0 C ; Experimental Assay: dG buffers:glycine or acetate: 5 mM or 10 mM, pH:2.5 ; Experimental Assay: dCp buffers:Sodium phosphate: 50 mM, pH:7.0 ; Experimental Assay: dHcal buffers:Sodium phosphate: 50 mM, pH:7.0 ; Experimental Assay: Tm buffers:Sodium phosphate: 50 mM, pH:7.0 ; Experimental Assay: dCp buffers:Sodium glycinate: 50 mM, pH:10.3 ; Experimental Assay: dHcal buffers:Sodium glycinate: 50 mM, pH:10.3 ; Experimental Assay: Tm buffers:Sodium glycinate: 50 mM, pH:10.3 ; Experimental Assay: dCp buffers:Sodium glycinate: 50 mM, pH:2.5 ; Experimental Assay: dHcal buffers:Sodium glycinate: 50 mM, pH:2.5 ; Experimental Assay: Tm buffers:Sodium glycinate: 50 mM, pH:2.5
Libraries Mutations for sequence ADKELKFLVVDDFSTMRRIVRNLLKELGFNNVEEAEDGVDALNKLQAGGYGFVISDWNMPNMDGLELLKTIRADGAMSALPVLMVTAEAKKENIIAAAQAGASGYVVKPFTAATLEEKLNKIFEKLGM

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 Chemotaxis protein CheY P0AE68 CHEY_ECO57
100.0 Chemotaxis protein CheY P0AE67 CHEY_ECOLI
100.0 Chemotaxis protein CheY P0AE69 CHEY_SHIFL
99.2 Chemotaxis protein CheY Q8FGP6 CHEY_ECOL6
97.7 Chemotaxis protein CheY P0A2D6 CHEY_SALTI
97.7 Chemotaxis protein CheY P0A2D5 CHEY_SALTY
94.6 Chemotaxis protein CheY Q9FAD7 CHEY_ENTCL
91.5 Chemotaxis protein CheY Q93P00 CHEY_YEREN
90.7 Chemotaxis protein CheY Q8D0P1 CHEY_YERPE