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 modification (Ca2+-myristoyl switch) by surface plasmon resonance spectroscopy. Either recoverin or phospholipid vesicles were immobilized on a sensor 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 myristoyl group. The Ca2+-myristoyl switch was half-maximal between 4.0 and 7.7 microM Ca2+, depending on whether recoverin or liposomes were in the mobile phase. At saturating [Ca2+], the dissociation constant (KD) of recoverin for phospholipid liposomes was approximately 150 microM. The association and dissociation of recoverin to membranes was fast and biphasic (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-membrane complex was 3-fold faster at low than at high free [Ca2+]. We discuss the analogy between the liposome-sensor chip and the disk surface 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+].
Lange
C
C
Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Germany.
Koch
K W
KW
D000818:Animals; D015374:Biosensing Techniques; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D005136:Eye Proteins; D051597:Hippocalcin; D007700:Kinetics; D008074:Lipoproteins; D008081:Liposomes; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D051603:Recoverin; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013057:Spectrum Analysis
D002021:Buffers; D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D008081:Liposomes; D009419:Nerve Tissue Proteins; D051603:Recoverin; D051597:Hippocalcin; D002118:Calcium