Abstract
N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Ca2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.
Highlights
Neuronal calcium sensors (NCSs) are EF-hand Ca2+-binding proteins responsible for transduction of calcium signals into a wide range of neuronal responses by regulating activity of effector enzymes and other target proteins
Five NCSs, namely recoverin, NCALD, guanylate cyclase activator protein 1 (GCAP1), guanylate cyclase activator protein 2 (GCAP2) and neuronal calcium sensor-1 (NCS-1), were expressed in bacteria according to the standard protocol and distribution of their myristoylated and non-myristoylated forms between soluble and insoluble fractions was studied
Recoverin was expressed mostly as a soluble protein and guanylate cyclase-activating proteins (GCAPs) were detected predominantly in the membrane fraction, whereas NCS-1 and NCALD were almost distributed between the supernatant and the pellet
Summary
Neuronal calcium sensors (NCSs) are EF-hand Ca2+-binding proteins responsible for transduction of calcium signals into a wide range of neuronal responses by regulating activity of effector enzymes and other target proteins (for review, see [1]). NCSs possess common domain architecture consisting of N-terminal and C-terminal domains, each containing two structurally coupled EF-hand motifs Among these motifs, only EF2 and EF3 (recoverin), EF3 and EF4 (KChIP1) or EF2, EF3 and EF4 (other KChIPs, GCAPs, VILIPs, NCS-1) are capable of binding calcium or magnesium, whereas EF1 is always non-functional due to the absence of certain amino acids responsible for metal ion coordination. NCSs are capable of binding zinc, which is suggested to be important for their normal and pathological activity [31,32]
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