Membrane binding of the neuronal calcium sensor recoverin - modulatory role of the charged carboxy-terminus.
BACKGROUND
The Ca2+-binding protein recoverin operates as a Ca2+-sensor in vertebrate photoreceptor cells. It undergoes a so-called Ca2+-myristoyl switch when cytoplasmic Ca2+-concentrations fluctuate in the cell. Its covalently attached myristoyl-group is exposed at high Ca2+-concentrations and enables recoverin to associate with lipid bilayers and to inhibit its target rhodopsin kinase. At low Ca2+-concentrations the myristoyl group is inserted into a hydrophobic pocket of recoverin thereby relieving inhibitory constraint on rhodopsin kinase. Hydrophobic and electrostatic interactions of recoverin with membranes have not been clearly determined, in particular the function of the positively charged carboxy-terminus in recoverin 191QKVKEKLKEKKL202 in this context is poorly understood.


RESULTS
Binding of myristoylated recoverin to lipid bilayer depends on the charge distribution in phospholipids. Binding was tested by equilibrium centrifugation and surface plasmon resonance (SPR) assays. It is enhanced to a certain degree by the inclusion of phosphatidylserine (up to 60%) in the lipid mixture. However, a recoverin mutant that lacked the charged carboxy-terminus displayed the same relative binding amplitudes as wildtype (WT) recoverin when bound to neutral or acidic lipids. Instead, the charged carboxy-terminus of recoverin has a significant impact on the biphasic dissociation of recoverin from membranes. On the other hand, the nonmyristoylated WT and truncated mutant form of recoverin did not bind to lipid bilayers to a substantial amount as binding amplitudes observed in SPR measurements are similar to bulk refractive index changes.


CONCLUSIONS
Our data indicate a small, but evident electrostatic contribution to the overall binding energy of recoverin association with lipid bilayer. Properties of the charged carboxy-terminus are consistent with a role of this region as an internal effector region that prolongs the time recoverin stays on the membrane by influencing its Ca2+-sensitivity.
Senin
Ivan I
II
Department of Biology and Environmental Sciences (Biochemistry group), University of Oldenburg, D-26111 Oldenburg, Germany. senin@belozersky.msu.ru
Churumova
Valeriya A
VA
Philippov
Pavel P
PP
Koch
Karl-Wilhelm
KW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D050356:Lipid Metabolism; D019814:Myristic Acid / Q000378:metabolism; D011485:Protein Binding; D051603:Recoverin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D020349:Surface Plasmon Resonance
D019814:Myristic Acid; D051603:Recoverin; D002118:Calcium