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FRET analysis of Calmodulin Binding to Nitric Oxide Synthase Peptides and Enzymes
Author(s) -
Spratt Donald E,
Taiakina Valentina,
Guillemette J Guy
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a645-b
Subject(s) - calmodulin , förster resonance energy transfer , chemistry , cofactor , nitric oxide synthase , enzyme , biochemistry , cysteine , biophysics , isozyme , alexa fluor , mutagenesis , gene isoform , fluorescence , mutant , biology , physics , quantum mechanics , gene
Calmodulin (CaM) is a ubiquitous Ca 2+ ‐sensor protein that binds and activates the Nitric Oxide Synthase (NOS) isozymes. Although the structure of CaM has been determined when bound to a peptide derived from the CaM‐binding domain of endothelial NOS, the conformation of CaM when bound to the three mammalian NOS holoenzymes has yet to be determined. Förster Resonance Energy Transfer (FRET) is a useful method for the determination of the distance between two different fluorescently labeled residues separated by ~10 to ~100 Å. The goal of our research is to determine the conformation of CaM when bound to all three NOS isoforms through the use of FRET. We have produced a double cysteine CaM mutant (T34C/T110C) using site‐directed mutagenesis for these FRET measurements. The NOS enzymes contain three cofactors (heme, FMN, and FAD) that absorb light strongly between 250 and 500 nm. In order to avoid undesired quenching, far‐red shifted fluorescent dyes (Alexa Fluor 546 C 5 ‐maleimide and DABMI) were chosen so that they can be selectively excited in the presence of the NOS cofactors. FRET studies were performed on the fluorescently labeled CaM with stoichiometric amounts of synthetic CaM‐binding domain NOS peptides and enzymes. These studies will provide a better understanding of the inherent differences between the Ca 2+ ‐dependent and Ca 2+ ‐independent binding of CaM to the mammalian NOS isozymes. This work is supported by NSERC.