Novel approaches to probe the binding of recoverin to membranes
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Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. We have investigated the interaction of recoverin with zwitterionic phosphatidylcholine bilayers, the major lipid component of the rod outer segment disk membranes, using both 31P and 19F solid-state nuclear magnetic resonance (NMR) and infrared spectroscopy. In particular, several novel approaches have been used, such as the centerband-only detection of exchange (CODEX) technique to investigate lipid lateral diffusion and 19F NMR to probe the environment of the recoverin myristoyl group. The results reveal that the lipid bilayer organization is not disturbed by recoverin. Non-myristoylated recoverin induces a small increase in lipid hydration that appears to be correlated with an increased lipid lateral diffusion. The thermal stability of recoverin remains similar in the absence or presence of lipids and Ca2+. Fluorine atoms have been strategically introduced at positions 4 or 12 on the myristoyl moiety of recoverin to, respectively, probe its behavior in the interfacial and more hydrophobic regions of the membrane. 19F NMR results allow the observation of the calcium–myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca2+ and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca2+.
KeywordsPhosphatidylcholine Infrared spectroscopy Solid-state nuclear magnetic resonance spectroscopy Lateral diffusion of lipids Multilamellar vesicles Fluorine
Centerband-only detection of exchange
Ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid
Fourier transform infrared
Full width at half maximum
High-performance liquid chromatography
Neuronal calcium sensor
Nuclear magnetic resonance
Recoverin with a fluorine atom at position 4 in the myristoyl moiety
Recoverin with a fluorine atom at position 12 in the myristoyl moiety
Rod outer segment
Gel-to-fluid phase transition temperature
Carbonyl stretching vibration
CH2 symmetric stretching vibration
This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (J.-F.P., C.S. and M.A.), the Fonds de recherche du Québec-Nature et Technologies (FRQ-NT) (J.-F.P. and M.A.), and Université Laval (J.-F.P., C.S. and M.A.). The Regroupement québécois de recherche sur la fonction, l’ingénierie et les applications des protéines (PROTEO), the Centre de recherche sur les matériaux avancés (CERMA), the Centre québécois sur les matériaux fonctionnels (CQMF), and the Centre de recherche du CHU de Québec are acknowledged for the infrastructure provided to us. PROTEO and CQMF are supported by FRQ-NT. K.P.-F. is the recipient of graduate scholarships from NSERC and FRQ-NT. G.V.-P. is the recipient of a graduate scholarship from the Fonds de recherche du Québec-Santé (FRQS). M.-C.G. is the recipient of graduate scholarships from PROTEO and NSERC. The authors would like to thank Jean-François Rioux-Dubé and François Paquet-Mercier for their support with the FT-IR experiments, and Matthieu Fillion for helpful discussions.
Compliance with Ethical Standards
Conflict of interest
The authors declare that there are no conflicts of interest.
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