Characterization of phospho-(tyrosine)-mimetic calmodulin mutants
Publisher
Public Library of ScienceDate
2015-04-01Citation
10.1371/journal.pone.0120798
Plos One 10.4 (2015): e0120798
ISSN
1932-6203DOI
10.1371/journal.pone.0120798Funded by
This work was funded by grants to AV from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494) and the Consejería de Educación de la Comunidad de Madrid (S2011/ BMD-2349), and grant to AV and DS from the European Commission (contract PITN-GA-2011- 289033 DYNANO). Additional funding to DS and MM was provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias. SRS was funded by a Marie Curie contract from the European Commission and VS and GB were supported by fellowship and grants (03-00-6057-2005 and PG-03-8728-2013) from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de VenezuelaProject
Comunidad de Madrid. S2010/BMD-2349/I2M2Editor's Version
http://dx.doi.org/10.1371/journal.pone.0120798Subjects
Calmodulin; Phospho-(Y)-mimetic CaM; Escherichia coli; MedicinaRights
© 2015 Stateva et al.Abstract
Calmodulin (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
Files in this item
Google Scholar:Stateva, Silviya R.
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Salas, Valentina
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Benaim, Gustavo
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Menéndez, Margarita
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Solís, Dolores
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Villalobo, Antonio
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