Solid-phase synthesis, mass spectrometric analysis of the zinc-folding, and phorbol ester-binding studies of the 116-mer peptide containing the tandem cysteine-rich C1 domains of protein kinase C gamma

Tumor-promoting phorbol esters activate protein kinase C (PKC) isozymes by binding to the zinc-finger like cysteine-rich domains in the N-terminal regulatory region. Our recent studies have revealed that only PKC gamma has two high affinity phorbol ester-binding domains, providing a structural blueprint for the rational design of PKC gamma-selective modulators for the treatment of neuropathic pain. To extend this approach, the 116-mer peptide containing the double cysteine-rich motifs of PKC gamma (gamma-C1A-C1B) has been synthesized for the first time using an Fmoc-solid phase strategy with a stepwise chain elongation. This peptide was purified by the reversed phase HPLC to give satisfactory mass data (MALDI-TOF-MS and ESI-TOF-MS). The peptide was successfully folded by zinc treatment and the folded peptide was analyzed intact under neutral conditions by ESI-TOF-MS. The multiple charge mass envelopes shifted to those of the lower mass charge state by addition of 4 molar equiv. ZnCl2, suggesting that gamma-C1A-C1B preserves some higher order structure by the zinc folding. Moreover, the mass spectrum of the zinc-folded peptide in the presence of EDTA dearly showed that gamma-C1A-C1B coordinates exactly four atoms of zinc. This zinc stoichiometry is identical to that of native PKC gamma. Scatchard analysis of the zinc-folded peptide revealed two binding sites of distinctly different affinities (Kd = 6.0 +/- 1.5 and 47.0 +/- 6.6 nM) comparable to those reported by Quest and Bell for the GST fusion protein of gamma-C1A-C1B prepared by DNA recombination. These results indicate that gamma-C1A-C1B serves as an effective surrogate for native PKC gamma for the study of the structural characteristics of the binding-recognition event and the design, discovery, and development of new PKC gamma-selective modulators. (C) 1999 Elsevier Science Ltd. All rights reserved.