title: Engineering an unnatural Nalpha-anchored disulfide into BPTI by total chemical synthesis: structural and functional consequences.
abstract: A disulfide-engineered analogue of bovine pancreatic trypsin inhibitor (BPTI), ((N(alpha)-(CH2)2S-)Gly38)BPTI, has been prepared using a thioester-mediated auxiliary functional group chemical ligation of a N(alpha)-ethanethiol-containing peptide segment with a peptide-alphaCOSR segment. In this study, Nalpha-(ethanethiol)Gly38 replaces the native Cys38, providing the sulfhydryl group required for ligation and folding. Comparisons between ((Nalpha-(CH2)2SH)Gly38)BPTI, synthetic native BPTI and reference BPTI purchased from Sigma were made using mass spectroscopy, enzyme inhibitor association constant determination (K(a)) and 1H-nuclear magnetic resonance total correlated spectroscopy (1H-NMR TOCSY) measurements. The K(a) value for ((Nalpha-(CH2)2SH)Gly38)BPTI was approximately 20-fold lower than synthetic and reference BPTI, which was attributed to perturbations in the binding loop of the protein (near Cys14). This hypothesis was confirmed by two-dimensional (2D) 1H-NMR TOCSY experiments. The data reported here demonstrate that total chemical synthesis by auxiliary functional group chemical ligation is a practical method for the synthesis of a novel class of biologically active protein analogues containing additional functional groups linked to the protein backbone.
authors.affiliation: The Scripps Research Institute, La Jolla, CA, USA. steveb@scripps.edu
authors.forename: S J
authors.initials: SJ
authors.lastname: Bark
authors.forename: S B
authors.initials: SB
authors.lastname: Kent
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000031:analogs & derivatives / Q000737:chemistry*; D002417:Cattle; D004220:Disulfides / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis; D014357:Trypsin / Q000737:chemistry
chemical_list: D004220:Disulfides; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D007611:Aprotinin; D014357:Trypsin