TY - JOUR
T1 - Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique
AU - Guerra-Castellano, Alejandra
AU - Díaz-Quintana, Antonio
AU - Moreno-Beltrán, Blas
AU - Lõpez-Prados, Javier
AU - Nieto, Pedro M.
AU - Meister, Wiebke
AU - Staffa, Jana
AU - Teixeira, Miguel
AU - Hildebrandt, Peter
AU - De La Rosa, Miguel A.
AU - Díaz-Moreno, Irene
PY - 2015/10/1
Y1 - 2015/10/1
N2 - 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.
AB - 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.
KW - cytochromes
KW - heme proteins
KW - NMR spectroscopy
KW - phosphorylation
KW - protein modifications
UR - http://www.scopus.com/inward/record.url?scp=84943814762&partnerID=8YFLogxK
U2 - 10.1002/chem.201502019
DO - 10.1002/chem.201502019
M3 - Article
AN - SCOPUS:84943814762
VL - 21
SP - 15004
EP - 15012
JO - Chemistry-A European Journal
JF - Chemistry-A European Journal
SN - 0947-6539
IS - 42
ER -
