Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647.
Recoverin belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. Ca2+-dependent conformational changes in recoverin are allosterically controlled by the covalently attached myristoyl group. The amino acid sequence of recoverin harbors a unique cysteine at position 38. The cysteine can be modified by the fluorescent dye Alexa647 using a maleimide-thiol coupling step. Introduction of Alexa647 into recoverin did not disturb the biological function of recoverin, as it can regulate rhodopsin kinase activity like unlabeled recoverin. Performance of the Ca2+-myristoyl switch of labeled recoverin was monitored by Ca2+-dependent association with immobilized lipids using surface plasmon resonance spectroscopy. When the Ca2+-concentration was varied, labeled myristoylated recoverin showed a 37%-change in fluorescence emission and a 34%-change in excitation intensity, emission and excitation maxima shifted by 6 and 18 nm, respectively. In contrast, labeled nonmyristoylated recoverin exhibited only minimal changes. Time-resolved fluorescence measurements showed biexponentiell fluorescence decay, in which the slower time constant of 2 ns was specifically influenced by Ca2+-induced conformational changes. A similar influence on the slower time constant was observed with the recoverin mutant RecE85Q that has a disabled EF-hand 2, but no such influence was detected with the mutant RecE121Q (EF-hand 3 is nonfunctional) that contains the myristoyl group in a clamped position. We conclude from our results that Alexa647 bound to cysteine 38 can monitor the conformational transition in recoverin that is under control of the myristoyl group.
Gensch
Thomas
T
IBI-1, Forschungszentrum Jülich, Jülich D-52425, Germany.
Komolov
Konstantin E
KE
Senin
Ivan I
II
Philippov
Pavel P
PP
Koch
Karl-Wilhelm
KW
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D019814:Myristic Acid / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D051603:Recoverin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
D005456:Fluorescent Dyes; C067574:RCV1 protein, Bos taurus; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D051603:Recoverin; C067297:8-aminohexylamino cAMP; D000242:Cyclic AMP; D051606:G-Protein-Coupled Receptor Kinase 1; D003545:Cysteine; D002118:Calcium