CALCIUM-DEPENDENT BINDING OF RECOVERIN TO MEMBRANES MONITORED BY SURFACE-PLASMON RESONANCE SPECTROSCOPY IN REAL-TIME

Recoverin is an N-myristoylated Ca2+-binding protein that serves as a Ca2+-sensor in visual transduction, We studied the dynamics of its Ca2 +-dependent membrane association which depends on the myristoyl modifi cation (Ca2+-myristoyl switch) by surface plasmon resonance spectrosco py. Either recoverin or phospholipid vesicles were immobilized on a se nsor chip surface, and the respective binding partner was supplied in the mobile phase, Binding of recoverin to artificial liposomes or rod outer segment membranes was strictly dependent on Ca2+ and the myristo yl group. The Ca2+-myristoyl switch was half-maximal between 4.0 and 7 .7 mu M Ca2+, depending on whether recoverin or liposomes were in the mobile phase. At saturating [Ca2+], the dissociation constant (K-D) of recoverin for phospholipid liposomes was approximately 150 mu M The a ssociation and dissociation of recoverin to membranes was fast and bip hasic (fast and slow components) with time constants on the order of 0 .1 s(-1) and 0.01 s(-1), respectively. Dissociation of the recoverin-m embrane complex was 3-fold faster at low than at high free [Ca2+]. We discuss the analogy between the liposome-sensor chip and the disk surf ace and conclude that a minor fraction of the total recoverin in a rod outer segment is associated with membranes at resting dark levels of free [Ca2+].