Detection of a concerted conformational change in the ATPase domain of DnaK triggered by peptide binding.
The molecular chaperone DnaK is composed of two functional domains, the ATPase domain and the substrate-binding domain. In this report, we show that peptide binding to DnaK can be sensed in real time through a labeled nucleotide bound in the ATPase domain. Specifically, when N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-triphosphate (MABA)-ATP.DnaK complexes are rapidly mixed with excess peptide, MABA fluorescence rapidly increases and the rate of increase is proportional to peptide concentration. Analysis of the formation traces yield on and off rate constants that are exactly equal to the rate constants obtained from experiments that directly probe peptide binding to DnaK. These results are the first to show that peptide binding to ATP.DnaK triggers a concerted conformational change in the ATPase domain.
Slepenkov
Sergey V
SV
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA.
Witt
Stephan N
SN
D000251:Adenosine Triphosphatases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010455:Peptides; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli