Interactions of peptides with DnaK and C-terminal DnaK fragments studied using fluorescent and radioactive peptides.
Monocysteine derivatives of Peptide C (KLIGVLSSLFRPK) were modified with N-((2-(acetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole (ANBD) to introduce a fluorescent probe. Five Peptide C derivatives-PepC-V5C-ANBD, PepC-L6C-ANBD, PepC-S7C-ANBD, PepC-S8C-ANBD, and PepC-L9C-ANBD-were then used to investigate the peptide-binding properties of DnaK. Introduction of the ANBD moiety at positions 8 and 9 of Peptide C yields peptides that bind to DnaK with a high affinity similar to unmodified peptide C. In contrast, the derivative carrying ANBD at position 6, PepC-L6C-ANBD, bound to DnaK with a binding affinity 470 times lower than that of PepC-L9C-ANBD. Peptide C derivatives carrying ANBD at positions 5 or 7 have intermediate DnaK binding affinities. By assaying the binding affinities of PepC-L9C-ANBD and PepNR-S6C-[1-14C]acetamide to DnaK and three C-terminal fragments of DnaK, DnaK 384-638 (residues 384 to 638), DnaK 389-607 (residues 389 to 607) and DnaK 386-561 (residues 386 to 561), we found that the last 31 residues of DnaK (residues 607-638) do not have a significant effect on the peptide binding to DnaK. However, residues 561 to 607, which form the C, D, and E alpha-helices directly adjacent to the peptide binding pocket of DnaK [X. Zhu, X. Zhou, W. F. Burkholder, A. Gragerov, C. M. Ogata, M. E. Gottesman, and W. A. Hendrickson, Science 272, 1606-1614, 1996], play important roles in stabilizing the DnaK/peptide complex. The kinetics of PepC-L9C-ANBD binding to DnaK, DnaK 384-638, DnaK 389-607, and DnaK 386-561 also support this observation.
Zhang
J
J
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, USA.
Walker
G C
GC
D001426:Bacterial Proteins / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D010069:Oxadiazoles / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D010069:Oxadiazoles; D010446:Peptide Fragments; D010455:Peptides; C050632:polypeptide C; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; C063842:dnaK protein, E coli; D003545:Cysteine