Chemical Protein Ubiquitylation with Preservation of the Native Cysteine Residues.
We report a cysteine-based ligation strategy for generating a monoubiquitylated protein while preserving the native cysteine residues on the acceptor protein. In monoubiquitylation of proliferating cell nuclear antigen (PCNA) this method circumvents the need to mutate the native cysteine residues on PCNA. The chemically ubiquitylated PCNA contains a noncleavable linkage of the same length as the native isopeptide linkage. It also retains the normal function of the native Ub-PCNA in stimulating the ATPase activity of replication factor C (RFC) and lesion bypass synthesis by Polη. This method may be adapted for chemical ubiquitylation of other proteins and for site-specific modification of a target protein at a specific site through sulfhydryl chemistry.
Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE, 19716, USA.
Kun
K
Yang
Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE, 19716, USA.
Guorui
G
Li
Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE, 19716, USA.
Ping
P
Gong
Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE, 19716, USA.
Weijun
W
Gui
Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE, 19716, USA.
Libo
L
Yuan
Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE, 19716, USA. zzhuang@udel.edu.
Zhihao
0000-0001-5047-9222
Z
Zhuang
D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D003545:Cysteine; D004259:DNA-Directed DNA Polymerase; D018809:Proliferating Cell Nuclear Antigen; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
caged cysteine; chemical ligation; photo-deprotection; protecting groups; protein modifications; ubiquitylation