Interactions of Peptides with DnaK and C-Terminal DnaK Fragments Studied Using Fluorescent and Radioactive Peptides

Monocysteine derivatives of Peptide C (KLIGVLSSLFRPK) were modified withN-((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 α-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,Science272, 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.