Engineering an unnatural Nα-anchored disulfide into BPTI by total chemical synthesis:: structural and functional consequences

A disulfide-engineered analogue of bovine pancreatic trypsin inhibitor (BPTI), ((N-alpha-(CH2)(2)S-)Gly(38))BPTI, has been prepared using a thioester-mediated auxiliary functional group chemical ligation of a N-alpha-ethanethiol-containing peptide segment with a peptide-(COSR)-C-alpha segment. In this study, N-alpha(ethanethiol)Gly(38) replaces the native Cys(38), providing the sulfhydryl group required for ligation and folding. Comparisons between ((N-alpha-(CH2)(2)SH)Gly(38))BPTI, synthetic native BPTI and reference BPTI purchased from Sigma were made using mass spectroscopy, enzyme inhibitor association constant determination (K-a) and H-1-nuclear magnetic resonance total correlated spectroscopy (H-1-NMR TOCSY) measurements. The K-a value for ((N-alpha(CH2)(2)SH)Gly(38))BPTI was approximately 20-fold lower than synthetic and reference BPTI, which was attributed to perturbations in the binding loop of the protein (near Cys(14)), This hypothesis was confirmed by two-dimensional (2D) H-1-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. (C) 1999 Federation of European Biochemical Societies.