title: Analysis of the phospholipase C-δ1 pleckstrin homology domain using native polyacrylamide gel electrophoresis.
abstract: The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP(3)) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP(3) binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP(3) and in thermal destabilization of the IP(3)-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP(3)-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP(3) and in thermal instability. These results indicated that the helical conformation of the α2-helix and the phenyl ring of Phe87 play important roles in the IP(3)-binding activity and thermal stability of the PLC-δ1 PH domain. Based on these results, the biological role of the α2-helix of the PLC-δ1 PH domain is discussed in terms of membrane binding.
authors.affiliation: Institute for Molecular Science, Myodaiji, Okazaki 444-8585, Japan.
authors.forename: Michikazu
authors.initials: M
authors.lastname: Tanio
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Nishimura
mesh_terms: D000818:Animals; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D063347:Native Polyacrylamide Gel Electrophoresis; D054803:Phospholipase C delta / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D008024:Ligands; D015544:Inositol 1,4,5-Trisphosphate; D054803:Phospholipase C delta