NUCLEAR-MAGNETIC-RESONANCE EVIDENCE FOR CA2-INDUCED EXTRUSION OF THE MYRISTOYL GROUP OF RECOVERIN()

Recoverin, a recently discovered member of the EF-hand protein superfa mily, serves as a Ca2+ sensor in vision, A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to translocate to retinal disc membranes when the Ca2+ level is ele vated. Two-dimensional H-1-C-13 shift correlation NMR spectra of recov erin containing a C-13-labeled myristoyl group were obtained to select ively probe the effect of Ca2+ on the environment of the attached myri stoyl group, In the Ca2+-free state, each pair of methylene protons bo nded to carbon atoms 2, 3, 11, and 12 of the myristoyl group gives ris e to two peaks. The splittings, caused by nonequivalent methylene prot on chemical shifts, indicate that the myristoyl group interacts intima tely with the protein in the Ca2+-free state. By contrast, only one pe ak is seen for each pair of methylene protons in the Ca2+-bound state, indicating that the myristoyl group is located in an isotropic enviro nment in this form. Furthermore, the H-1-C-13 shift correlation NMR sp ectrum of Ca2+-bound recoverin is very similar to that of myristic aci d in solution, H-1-C-13 Shift correlation NMR experiments were also pe rformed with C-13-labeled recoverin to selectively probe the resonance s of methyl groups in the hydrophobic core of the protein. The spectru m of Ca2+-bound myristoylated recoverin is different from that of Ca2-free myristoylated recoverin but similar to that of Ca2+-bound unmyri stoylated recoverin. Hence, the myristoyl group interacts little with the hydrophobic core of myristoylated recoverin in the Ca2+-bound stat e. Three-dimensional (C-13/F-1)-edited (C-13/F-3)-filtered heteronucle ar multiple quantum correlation-nuclear Overhauser effect spectroscopy spectra of recoverin containing a C-13-labeled myristoyl group were o btained to selectively probe protein residues located within 5 Angstro m of the myristoyl group. The myristoyl group makes close contact with a number of aromatic residues in Ca2+-free recoverin, whereas the myr istoyl group makes no observable contacts with the protein in the Ca2-bound state. These NMR data demonstrate that the binding of Ca2+ to r ecoverin induces the extrusion of its myristoyl group into the solvent , which would enable it to interact with a lipid bilayer or a hydropho bic site of a target protein.