CALCIUM-DEPENDENT SOLVATION OF THE MYRISTOYL GROUP OF RECOVERIN

Recoverin is an N-myristoylated calcium-binding protein present in the photoreceptor cells of the mammalian retina. It is believed to functi on as a calcium sensor in visual signal transduction by coupling the k inetics of the recovery phase of the photoresponse to changes in the l evels of intracellular Ca2+. Upon binding Ca2+, recoverin undergoes a conformational change that allows it to associate with membranes in a manner that requires N-myristoyl modification. It has been proposed th at, in the Ca2+-free conformation, the myristoyl group is sequestered in a hydrophobic part of the protein, and in the Ca2+-bound conformati on, the myristoyl group is exposed to solution. The crystal structure of Ca2+-bound recoverin reveals an exposed cluster of hydrophobic resi dues, raising the possibility that residues in this region may functio n as part of an intramolecular myristoyl binding site. Fluorescence sp ectroscopy analysis of interactions between recoverin and 1-anilinonap hthalene-8-sulfonate (ANS) shows that an increase in solvent-accessibl e hydrophobic surface accompanies Ca2+ binding. H-1 nuclear magnetic r esonance (NMR) spectra of myristoyl protons show dispersed chemical sh ifts in the Ca2+-free conformation that become relatively uniform upon the addition of Ca2+. Two-dimensional nuclear Overhauser effect (NOE) spectra of Ca2+-free recoverin show NOE contacts between myristoyl pr otons and aromatic ring proximity to a tryptophan residue only in the Ca2+-free conformation. These results indicate that the myristoyl grou p is in contact with residues in the hydrophobic cluster in Ca2+-free recoverin and that it is exposed to solution in the Ca2+-bound conform ation.