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 PKCgamma has two high affinity phorbol ester-binding domains, providing a structural blueprint for the rational design of PKCgamma-selective modulators for the treatment of neuropathic pain. To extend this approach, the 116-mer peptide containing the double cysteine-rich motifs of PKCgamma (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 clearly showed that gamma-C1A-C1B coordinates exactly four atoms of zinc. This zinc stoichiometry is identical to that of native PKCgamma. 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 PKCgamma for the study of the structural characteristics of the binding recognition event and the design, discovery, and development of new PKCgamma-selective modulators.
Fukuda
H
H
Nihon PerSeptive Ltd., Tokyo, Japan.
Irie
K
K
Nakahara
A
A
Ohigashi
H
H
Wender
P A
PA
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011487:Protein Conformation; D015202:Protein Engineering; D011493:Protein Kinase C / Q000737:chemistry*; D014316:Tritium; D016335:Zinc Fingers
D007527:Isoenzymes; D010455:Peptides; D014316:Tritium; D015240:Phorbol 12,13-Dibutyrate; C087857:protein kinase C gamma; D011493:Protein Kinase C; D003545:Cysteine