Interactions of Peptides with DnaK and C-Terminal DnaK Fragments Studied Using Fluorescent and Radioactive Peptides Jundong Zhang 1 and Graham C. Walker 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received March 2, 1998, and in revised form May 26, 1998 Monocysteine derivatives of Peptide C (KLIGVLSS- LFRPK) 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 deriva- tives—PepC-V5C-ANBD, PepC-L6C-ANBD, PepC-S7C- ANBD, PepC-S8C-ANBD, and PepC-L9C-ANBD—were then used to investigate the peptide-binding proper- ties of DnaK. Introduction of the ANBD moiety at po- sitions 8 and 9 of Peptide C yields peptides that bind to DnaK with a high affinity similar to unmodified pep- tide 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 affin- ities. By assaying the binding affinities of PepC-L9C- ANBD and PepNR-S6C-[1- 14 C]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 -helices directly adjacent to the peptide binding pocket of DnaK [X. Zhu, X. Zhou, W. F. Burk- holder, 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 com- plex. The kinetics of PepC-L9C-ANBD binding to DnaK, DnaK 384 – 638, DnaK 389 – 607, and DnaK 386 – 561 also support this observation. © 1998 Academic Press Key Words: DnaK; Peptide C; Peptide NR; peptide binding; fluorescent peptides. DnaK protein of Escherichia coli is a well-character- ized member of the family of highly conserved heat- shock proteins with a molecular mass of 70 kDa (Hsp70) 3 (1, 2). Working in conjunction with DnaJ and GrpE as part of a molecular chaperone machine, DnaK plays a variety of physiological roles by functioning as a molecular chaperone (3). DnaK, like other members of the Hsp70 family, consists of a highly conserved N-terminal domain that encodes a weak ATPase activ- ity, a peptide-binding domain that is directly adjacent to the ATPase domain, and an extreme C-terminal region (4 – 6). Like other Hsp70s, the ATPase domain of DnaK and the peptide-binding domain of DnaK are functionally coupled, and this coupling of the domains of DnaK is essential for the biological functions of DnaK (7, 8). The interaction of ATP with the N-termi- nal domain of DnaK induces a conformational change in the peptide-binding domain of DnaK and leads to peptide release, whereas the binding of peptides to the peptide-binding domain of DnaK affects the conforma- tion of the N-terminal domain and stimulates its AT- Pase activity (9 –11). Phage-displayed peptide libraries and synthetic pep- tide libraries have been used to study the substrate specificity of DnaK (5) and other Hsp70s (12–14). These studies have suggested that peptides composed of hydrophobic amino acids are likely to have high affinity to DnaK and other Hsp70s. The high selectivity 1 Present address: RepliGen Corporation, 117 4th Avenue, Need- ham, MA 02194. 2 To whom correspondence should be addressed at 68-633, Depart- ment of Biology, Massachusetts Institute of Technology, 77 Massa- chusetts Avenue, Cambridge, MA 02139. Fax: 617-253-2643. 3 Abbreviations used: Hsp70, heat-shock protein with a molecular mass of 70 kDa; PepNR, Peptide NR; I-ANBD, N-((2-(iodoacetoxy)- ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; TFA, trifluo- roacetic acid; I-ABASA, N-iodoacetamidobutyl-4-aziobsalicylic acid. 0003-9861/98 $25.00 177 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 356, No. 2, August 15, pp. 177–186, 1998 Article No. BB980784