%0 Journal Article
%A Gensch, T.
%A Komolov, K. E.
%A Senin, I. I.
%A Philippov, P. P.
%A Koch, K.-W.
%T Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647
%J Proteins
%V 66
%@ 0887-3585
%C New York, NY
%I Wiley-Liss
%M PreJuSER-56498
%P 492 - 499
%D 2007
%Z Record converted from VDB: 12.11.2012
%X 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.
%K Amino Acid Substitution
%K Animals
%K Calcium: pharmacology
%K Calcium: physiology
%K Cattle
%K Cyclic AMP: analogs & derivatives
%K Cyclic AMP: chemistry
%K Cysteine: chemistry
%K Fluorescent Dyes: chemistry
%K G-Protein-Coupled Receptor Kinase 1: metabolism
%K Models, Molecular
%K Mutagenesis, Site-Directed
%K Mutation, Missense
%K Myristic Acid: chemistry
%K Point Mutation
%K Protein Binding
%K Protein Conformation
%K Protein Processing, Post-Translational
%K Protein Structure, Tertiary
%K Recombinant Fusion Proteins: chemistry
%K Recoverin: chemistry
%K Recoverin: drug effects
%K Recoverin: genetics
%K Spectrometry, Fluorescence
%K Structure-Activity Relationship
%K Surface Plasmon Resonance
%K Fluorescent Dyes (NLM Chemicals)
%K RCV1 protein, Bos taurus (NLM Chemicals)
%K Recombinant Fusion Proteins (NLM Chemicals)
%K Recoverin (NLM Chemicals)
%K 8-aminohexylamino cAMP (NLM Chemicals)
%K Cysteine (NLM Chemicals)
%K Myristic Acid (NLM Chemicals)
%K Cyclic AMP (NLM Chemicals)
%K Calcium (NLM Chemicals)
%K G-Protein-Coupled Receptor Kinase 1 (NLM Chemicals)
%K J (WoSType)
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:17078090
%U <Go to ISI:>//WOS:000243358000018
%R 10.1002/prot.21231
%U https://juser.fz-juelich.de/record/56498