title: Visualization of a slow, ATP-induced structural transition in the bacterial molecular chaperone DnaK.
abstract: Recent reports have shown that the binding of ATP to a 70-kDa molecular chaperone induces a rapid global conformational transition from a "high affinity" state to a "low affinity" state, where these states are defined by tight and weak binding to (poly)peptides, respectively. To complete the activity cycle, a chaperone molecule must ultimately return to the high affinity state. In this report, this return to the high affinity state was studied using a chemical cross-linking assay in conjunction with SDS-polyacrylamide gel electrophoresis. The basis for this assay is that in the absence of nucleotide or in the presence of ADP, conditions that stabilize the high affinity state, cross-linking of the Escherichia coli molecular chaperone DnaK yielded two monomeric forms, with apparent molecular masses of 70 kDa (77%) and 90 kDa (23%), whereas cross-linking yielded only the 70-kDa monomeric form in the presence of ATP. This ATP-dependent difference in cross-linking was used to follow the kinetics of the low affinity to high affinity transition under single turnover conditions. The rate of this transition (kobs = 3.4 (+/-0.6) x 10(-4) s-1 at 25 degrees C) is almost identical to the reported rate of ATP hydrolysis (khy = 2.7 (+/-0.7) x 10(-4) s-1 at 22 degrees C). These results are consistent with a two-step sequential reaction where rate-limiting ATP hydrolysis precedes the conformational change. Models for the formation of two cross-linked DnaK monomers in the absence of ATP are discussed.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Farr
authors.forename: S V
authors.initials: SV
authors.lastname: Slepenkov
authors.forename: S N
authors.initials: SN
authors.lastname: Witt
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli