Nov 30, 1993

Thermodynamic analysis of the chemotactic protein from Escherichia coli, CheY

Biochemistry
V V FilimonovL Serrano

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...Continue Reading

  • References37
  • Citations32

Citations

Mentioned in this Paper

Thermodynamics
Bacterial Proteins
Alkalescens-Dispar Group
Proteins, Recombinant DNA
Tertiary Protein Structure
Sequence Determinations
Fluorescence Spectroscopy
Ampholytes
Mass Spectrometry
Calorimetry, Differential Scanning

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