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Structure, Topology, and Dynamics of Myristoylated Recoverin Bound to Phospholipid Bilayers

2003; American Chemical Society; Volume: 42; Issue: 21 Linguagem: Inglês

10.1021/bi0206816

1943-295X

Kathleen G. Valentine, Michael F. Mesleh, Stanley J. Opella, Mitsuhiko Ikura, James B. Ames,

Neuroscience and Neuropharmacology Research

Recoverin, a member of the EF-hand protein superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl group covalently attached to the N-terminus of recoverin facilitates its binding to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Samples of 15N-labeled Ca2+-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis of 15N and 31P chemical shifts observed in solid-state NMR spectra. On the basis of a 2H NMR order parameter analysis performed on recoverin containing a fully deuterated myristoyl group, the N-terminal myristoyl group appears to be located within the lipid bilayer. Two-dimensional solid-state NMR (1H−15N PISEMA) spectra of uniformly and selectively 15N-labeled recoverin show that the Ca2+-bound protein is positioned on the membrane surface such that its long molecular axis is oriented ∼45° with respect to the membrane normal. The N-terminal region of recoverin points toward the membrane surface, with close contacts formed by basic residues K5, K11, K22, K37, R43, and K84. This orientation of the membrane-bound protein allows an exposed hydrophobic crevice, near the membrane surface, to serve as a potential binding site for the target protein, rhodopsin kinase. Close agreement between experimental and calculated solid-state NMR spectra of recoverin suggests that membrane-bound recoverin retains the same overall three-dimensional structure that it has in solution. These results demonstrate that membrane binding by recoverin is achieved primarily by insertion of the myristoyl group inside the bilayer with apparently little rearrangement of the protein structure.