Circular dichroism and 1H nuclear magnetic resonance studies on the solution and membrane structures of GAP-43 calmodulin-binding domain.

Growth-associated protein-43 (GAP-43) is believed to be palmitoylated near the N terminus and the modification is assumed to be involved in the membrane anchoring of the protein. However, GAP-43 isolated from bovine brain is not palmitoylated as shown by mass spectrometric analysis, but still retains the ability to bind phospholipids, suggesting that other parts of the molecule are involved in the interaction. Upon addition of acidic phospholipids, purified GAP-43 showed a conformational change from random coil to alpha-helix as indicated by a change in CD spectra. A synthetic peptide corresponding to the calmodulin-binding domain showed a similar conformational change from random coil to alpha-helix in the presence of various acidic phospholipids. These results suggest that the calmodulin-binding domain of GAP-43 is directly involved in the GAP-43-membrane interaction and undergoes a conformational change upon binding to phospholipid membranes. After phosphorylation by protein kinase C, the phospholipid-induced conformational changes were no longer observed. Structural characteristics of the calmodulin-binding domain peptide in aqueous and hydrophobic solvents were further studied in detail by two-dimensional 1H nuclear magnetic resonance. The results obtained suggest that the domain assumes a nascent alpha-helical structure in aqueous solution, which is stabilized under hydrophobic environments.