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| Hauptseite > Publikationsdatenbank > Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647 |
| Hauptseite > Publikationsdatenbank > Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647 |
| Journal Article | PreJuSER-56498 |
; ; ; ;
2007
Wiley-Liss
New York, NY
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Please use a persistent id in citations: doi:10.1002/prot.21231
Abstract: 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.
Keyword(s): Amino Acid Substitution (MeSH) ; Animals (MeSH) ; Calcium: pharmacology (MeSH) ; Calcium: physiology (MeSH) ; Cattle (MeSH) ; Cyclic AMP: analogs & derivatives (MeSH) ; Cyclic AMP: chemistry (MeSH) ; Cysteine: chemistry (MeSH) ; Fluorescent Dyes: chemistry (MeSH) ; G-Protein-Coupled Receptor Kinase 1: metabolism (MeSH) ; Models, Molecular (MeSH) ; Mutagenesis, Site-Directed (MeSH) ; Mutation, Missense (MeSH) ; Myristic Acid: chemistry (MeSH) ; Point Mutation (MeSH) ; Protein Binding (MeSH) ; Protein Conformation (MeSH) ; Protein Processing, Post-Translational (MeSH) ; Protein Structure, Tertiary (MeSH) ; Recombinant Fusion Proteins: chemistry (MeSH) ; Recoverin: chemistry (MeSH) ; Recoverin: drug effects (MeSH) ; Recoverin: genetics (MeSH) ; Spectrometry, Fluorescence (MeSH) ; Structure-Activity Relationship (MeSH) ; Surface Plasmon Resonance (MeSH) ; Fluorescent Dyes ; RCV1 protein, Bos taurus ; Recombinant Fusion Proteins ; Recoverin ; 8-aminohexylamino cAMP ; Cysteine ; Myristic Acid ; Cyclic AMP ; Calcium ; G-Protein-Coupled Receptor Kinase 1 ; J ; EF-hand (auto) ; Ca2+-binding protein (auto) ; myristoyl switch (auto) ; fluorescence emission and excitation (auto)
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