Characterization of two partially unfolded intermediates of the molecular chaperone DnaK at low pH.
In this study, the effect of pH on the conformation and the reactivity of the Escherichia coli Hsp70 molecular chaperone DnaK was investigated using spectroscopic and chemical assays. DnaK exhibits negligible binding of the hydrophobic dye 1-anilino-naphthalene-8-sulfonate (ANS) between pH 7 to 5.0, whereas appreciable binding occurs between pH 4.5 to 4.0. The binding of ANS to a protein is diagnostic of the presence of accessible ordered hydrophobic surfaces. Such hydrophobic surfaces are often displayed by partially folded protein intermediates such as molten globules. Nucleotide inhibits 70% of the ANS binding at pH 4.5 but none of the ANS binding at pH 4.0. Proteolysis of nucleotide-free DnaK at pH 4.5 with cathepsin D yields detectable fragments (masses > 20 kDa) of the C-terminal peptide-binding domain but none of the N-terminal ATPase domain, thus the ATPase domain is preferentially targeted for proteolysis. In contrast, proteolysis of nucleotide-free DnaK at pH 4.0 with cathepsin D cuts near the linker region, yielding both functional domains. Our interpretation of these data is that incubation of DnaK at pH 4.5 produces a partially unfolded form of the ATPase domain, in which secondary structure is mainly intact, but tertiary structure is reduced. Incubation of the protein at pH 4.0 produces an intermediate in which both functional domains have collapsed and possibly separated. Nucleotide inhibits the conformational change that occurs at pH 4.5 but not at 4.0.
Sehorn
Michael G
MG
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932, USA.
Slepenkov
Sergey V
SV
Witt
Stephan N
SN
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D002151:Calorimetry; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli