Selective photoaffinity labeling of the inositol polyphosphate binding C2B domains of synaptotagmins.
Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca2+ sensor and provides both phospholipid and inositol polyphosphate (IPn) recognition domains important in endo- and exocytosis. Four photoaffinity analogues of IP3, IP4, and IP6 containing a P-1- or P-2-linked 4-benzoyldihydrocinnamidyl (BZDC) photophore were used to label glutathione S-transferase (GST) fusion constructs of the Syt II-C2A and C2B domains. The P-2-linked [3H]BZDC-IP6 showed efficient, IP6-displaceable labeling of the GST-Syt II-C2B. The rank order of photocovalent modification paralleled the order of competitive displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [3H]BZDC-IP6 failed to label the C2B domains. The GST-Syt III-C2B domain, which lacks IP6 binding affinity, also failed to undergo labeling by P-2-linked [3H]BZDC-IP6. When mixtures of the 32-amino acid basic peptide corresponding to the essential IPn binding region of the Syt II-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amount of P-2-linked [3H]BZDC-IP6, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP6 appeared to induce a binding-compatible structure in the short peptide.
Mehrotra
B
B
Departments of Chemistry, Biochemistry and Cell Biology, University at Stony Brook, Stony Brook, New York 11794-3400, USA.
Elliott
J T
JT
Chen
J
J
Olszewski
J D
JD
Profit
A A
AA
Chaudhary
A
A
Fukuda
M
M
Mikoshiba
K
K
Prestwich
G D
GD
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000737:chemistry; D001667:Binding, Competitive; D007295:Inositol Phosphates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D050861:Synaptotagmin II; D014316:Tritium
D000345:Affinity Labels; D001577:Benzophenones; D007295:Inositol Phosphates; D009419:Nerve Tissue Proteins; D010455:Peptides; D050861:Synaptotagmin II; C494263:Syt2 protein, mouse; D014316:Tritium