Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique.
Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe-Met bond in the ferric form of cytochrome c, thereby lowering the pKa value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.
IBVF - cicCartuja, Universidad de Sevilla - CSIC, Avenida Américo Vespucio 49, Sevilla 41092 (Spain).
Alejandra
A
Guerra-Castellano
IBVF - cicCartuja, Universidad de Sevilla - CSIC, Avenida Américo Vespucio 49, Sevilla 41092 (Spain). qzaida@us.es.
Antonio
A
Díaz-Quintana
IBVF - cicCartuja, Universidad de Sevilla - CSIC, Avenida Américo Vespucio 49, Sevilla 41092 (Spain).
Blas
B
Moreno-Beltrán
IIQ - cicCartuja, Universidad de Sevilla - CSIC, Avenida Américo Vespucio 49, Sevilla 41092 (Spain).
Javier
J
López-Prados
IIQ - cicCartuja, Universidad de Sevilla - CSIC, Avenida Américo Vespucio 49, Sevilla 41092 (Spain).
Pedro M
PM
Nieto
Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Strasse des 17. Juni 135, 10623 Berlin (Germany).
Wiebke
W
Meister
Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Strasse des 17. Juni 135, 10623 Berlin (Germany).
Jana
J
Staffa
Instituto de Tecnologia Química e Biológica, António Xavier Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras (Portugal).
Miguel
M
Teixeira
Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Strasse des 17. Juni 135, 10623 Berlin (Germany).
Peter
P
Hildebrandt
IBVF - cicCartuja, Universidad de Sevilla - CSIC, Avenida Américo Vespucio 49, Sevilla 41092 (Spain).
Miguel A
MA
De la Rosa
IBVF - cicCartuja, Universidad de Sevilla - CSIC, Avenida Américo Vespucio 49, Sevilla 41092 (Spain). idiazmoreno@us.es.
Irene
I
Díaz-Moreno
NMR spectroscopy; cytochromes; heme proteins; phosphorylation; protein modifications