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 f...

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Published in:Chemistry : a European journal 2015-10, Vol.21 (42), p.15004-15012
Main Authors: Guerra-Castellano, Alejandra, Díaz-Quintana, Antonio, Moreno-Beltrán, Blas, López-Prados, Javier, Nieto, Pedro M., Meister, Wiebke, Staffa, Jana, Teixeira, Miguel, Hildebrandt, Peter, De la Rosa, Miguel A., Díaz-Moreno, Irene
Format: Article
Language:English
Subjects:
cytochromes
heme proteins
NMR spectroscopy
phosphorylation
protein modifications
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Summary: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. An efficient site‐specific incorporation of the non‐canonical amino acid p‐carboxymethyl‐L‐phenylalanine (pCMF) into human cytochrome c using the evolved tRNA approach is demonstrated (see figure). This allows the mimicking of tyrosine phosphorylation, which is related to the onset of a wide range of human diseases. Moreover, an optimized synthesis of pCMF allows its production in improved yield at reduced cost.
ISSN:0947-6539
1521-3765
DOI:10.1002/chem.201502019