title: DnaK, hsp73, and their molten globules. Two different ways heat shock proteins respond to heat.
abstract: The thermal stability of bovine brain hsp73, Escherichia coli DnaK, and its mutant T199A was studied by a combination of spectroscopic and chromatographic methods. DnaK undergoes a temperature-induced conformational change that leads to the formation of a molten globule at physiologically relevant temperatures (midpoint of the transition, tm, 41 degrees C). Native DnaK binds to a denatured form of alpha-lactalbumin in a temperature-dependent manner with maximum rate at about 40 degrees C. The molten globule of DnaK is unable to bind denatured alpha-lactalbumin but recovers native structure and activity upon cooling. The half-life of the refolding process is 10 min at 35 degrees C. Mg/ATP and Mg/ADP increase the thermal stability of DnaK; in the presence of these nucleotides the tm is shifted to 59 degrees C. Binding of Mg/ATP (but not Mg/ADP or Mg/adenosine 5'-[gamma-thio]triphosphate) causes a conformational change in DnaK as determined by the emission fluorescence spectrum. The DnaK mutant T199A which lacks the threonine residue that is essential for ATP hydrolysis and autophosphorylation activity (McCarty, J. S., and Walker, G. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9513-9517) shows nearly identical properties to the wild type in the presence or absence of nucleotides. Hsp73 undergoes similar temperature-induced transitions as determined by spectroscopic methods (Palleros, D.R., Welch, W.J., and Fink, A.L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5719-5723); however, contrary to DnaK, the molten globule of hsp73 irreversibly aggregates at temperatures higher than its tm (42 degrees C).
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
authors.forename: D R
authors.initials: DR
authors.lastname: Palleros
authors.forename: K L
authors.initials: KL
authors.lastname: Reid
authors.forename: J S
authors.initials: JS
authors.lastname: McCarty
authors.forename: G C
authors.initials: GC
authors.lastname: Walker
authors.forename: A L
authors.initials: AL
authors.lastname: Fink
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D007768:Lactalbumin / Q000378:metabolism; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007768:Lactalbumin; C063842:dnaK protein, E coli