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Title

Characterization of phospho-(tyrosine)-mimetic calmodulin mutants

AuthorsStateva, Silvia R.; Salas, Valentina; Benaim, Gustavo; Menéndez, Margarita ; Solís, D. ; Villalobo, Antonio
Issue Date2015
PublisherPublic Library of Science
CitationPLoS ONE 10(4): e0120798 (2015)
AbstractCalmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro.
DescriptionThis is an open access article distributed under the terms of the Creative Commons Attribution License.
Publisher version (URL)http://dx.doi.org/10.1371/journal.pone.0120798
URIhttp://hdl.handle.net/10261/124795
DOI10.1371/journal.pone.0120798
Identifiersdoi: 10.1371/journal.pone.0120798
issn: 1932-6203
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