STRUCTURAL CHARACTERIZATION OF RECOVERIN, CALCIUM BINDING PROTEIN IN VISION

University Of Pennsylvania, Philadelphia PA

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Abstract

Recoverin is a 23 kD, N-myristoylated, calcium binding protein found in vertebrate photoreceptor cells. It is involved in the recovery phase of visual excitation and in adaptation to background light. It influences the photoresponse by blocking the phosphorylation of photoexcited rhodopsin. The N-myristoylation of recoverin is found to play a key role in the Ca+ mediated structural switch of the protein. This calcium-myristoyl switch enables the translocation of recoverin from the cytosol to the membrane where it imparts its functioning in the regulatory cascade of the retinal rod cells. The crystal structure of unmyristoylated recoverin, with a single Ca+ bound can be compared to the solution NMR structure determined on the myristoylated Ca+ free form of the protein. Both structures show a similar overall fold containing 4 EF-hand motifs. There is a variation in the helix crossing axis of the two forms of the protein. This structural change can provide information regarding the molecular mechanism of the calcium-myristoyl switch. The initial solid-state NMR studies of myristoylated recoverin in the Ca+ bound form indicate the presence of the EF- hand helices in a topology that lies perpendicular to the bilayer normal. The two-dimensional PISEMA spectrum of uniformly 15N labeled myristoylated recoverin in dimyristoyl phosphocholine (DMPC) bilayers indicates the majority of resonance intensity in the in-plane helical region of the 15N chemical shift spectrum. It demonstrates that the protein can be mechanically oriented in bilayers on glass plates with the myristoyl chain anchoring the protein in the bilayer. 31P NMR spectra confirm the orientation of the bilayer with respect to the magnetic field. Further studies will be conducted to calculate the three-dimensional structure of recoverin inserted in lipid bilayers to determine the crossing axes of the EF-hand helices.