SELECTIVE PHOTOAFFINITY-LABELING OF THE INOSITOL POLYPHOSPHATE BINDING C2B DOMAINS OF SYNAPTOTAGMINS

Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copi es of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca2+ sensor and provides both phospholi pid and inositol polyphosphate (IPn) recognition domains important in endo- and exocytosis. Four photoaffinity analogues of IP3, IP4, and IP 6 containing a P-1- or P-2-linked 4-benzoyldihydrocinnamidyl (BZDC) ph otophore were used to label glutathione S-transferase (GST) fusion con structs of the Syt II-C2A and C2B domains. The P-2-linked [H-3]BZDC-IP 6 showed efficient, IP6-displaceable labeling of the GST-Syt II-C2B. T he rank order of photocovalent modification paralleled the order of co mpetitive displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [ H-3]BZDC-IP6 failed to label the C2B domains. The GST-Syt III-C2B doma in, which lacks IP6 binding affinity, also failed to undergo labeling by P-2-linked [H-3]BZDC-IP6. When mixtures of the 32-amino acid basic peptide corresponding to the essential IPn binding region of the Syt I I-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amoun t of P-2-linked [H-3]BZDC-IP6, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer a t two pH values showed a random coil, the photoaffinity analogue of IP 6 appeared to induce a binding-compatible structure in the short pepti de.