TY  - JOUR
AU  - Gensch, T.
AU  - Komolov, K. E.
AU  - Senin, I. I.
AU  - Philippov, P. P.
AU  - Koch, K.-W.
TI  - Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647
JO  - Proteins
VL  - 66
SN  - 0887-3585
CY  - New York, NY
PB  - Wiley-Liss
M1  - PreJuSER-56498
SP  - 492 - 499
PY  - 2007
N1  - Record converted from VDB: 12.11.2012
AB  - 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.
KW  - Amino Acid Substitution
KW  - Animals
KW  - Calcium: pharmacology
KW  - Calcium: physiology
KW  - Cattle
KW  - Cyclic AMP: analogs & derivatives
KW  - Cyclic AMP: chemistry
KW  - Cysteine: chemistry
KW  - Fluorescent Dyes: chemistry
KW  - G-Protein-Coupled Receptor Kinase 1: metabolism
KW  - Models, Molecular
KW  - Mutagenesis, Site-Directed
KW  - Mutation, Missense
KW  - Myristic Acid: chemistry
KW  - Point Mutation
KW  - Protein Binding
KW  - Protein Conformation
KW  - Protein Processing, Post-Translational
KW  - Protein Structure, Tertiary
KW  - Recombinant Fusion Proteins: chemistry
KW  - Recoverin: chemistry
KW  - Recoverin: drug effects
KW  - Recoverin: genetics
KW  - Spectrometry, Fluorescence
KW  - Structure-Activity Relationship
KW  - Surface Plasmon Resonance
KW  - Fluorescent Dyes (NLM Chemicals)
KW  - RCV1 protein, Bos taurus (NLM Chemicals)
KW  - Recombinant Fusion Proteins (NLM Chemicals)
KW  - Recoverin (NLM Chemicals)
KW  - 8-aminohexylamino cAMP (NLM Chemicals)
KW  - Cysteine (NLM Chemicals)
KW  - Myristic Acid (NLM Chemicals)
KW  - Cyclic AMP (NLM Chemicals)
KW  - Calcium (NLM Chemicals)
KW  - G-Protein-Coupled Receptor Kinase 1 (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:17078090
UR  - <Go to ISI:>//WOS:000243358000018
DO  - DOI:10.1002/prot.21231
UR  - https://juser.fz-juelich.de/record/56498
ER  -