Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary.
We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet(7)]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the alpha-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximately 45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.
Low
D W
DW
Gryphon Sciences, South San Francisco, CA 94080; and Department of Chemistry, Occidental College, Los Angeles, CA 90041, USA. dlow@gryphonsci.com
Hill
M G
MG
Carrasco
M R
MR
Kent
S B
SB
Botti
P
P
D000595:Amino Acid Sequence; D003573:Cytochrome b Group / Q000138:chemical synthesis* / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; C017850:cytochrome b562, E coli