Isomorphous replacement of cystine with selenocystine in endothelin: Oxidative refolding, biological and conformational properties of [Sec(3),Sec(11),Nle(7)]-endothelin-1

Air re-oxidation of fully reduced human endothelin-1 under optimized condit ions yields the natural isomer with parallel disulfide bridges and the non- natural isomer with crossed disulfide bridges at a ratio of 3:1. in view of die recently determined highly reducing redox potential of selenocysteine (-381 mV) in peptides, the half-cystine residues Cys(3) and Cys(11) of the natural isomer of ondothelin-1 were replaced by selenocysteine. Taking adva ntage of the high stability of the diselenide group toward reducing agents for disulfides a regioselective disulfide bridging of the second cysteine p air allowed for straightforward preparation of the [Sec(3),Sec(11),Nle(7)]- endothelin-1. NMR structural analysis showed conformational preferences of this endothelin analog that were identical to those of the natural hormone. Similarly, the bioactivity data confirmed that replacement of cysteine res idues with selenocysteine was without detectable effect on receptor recogni tion and signal transduction. Both findings strongly support that die excha nge of sulfur against selenium produces a fully isomorphous molecule as rec ently observed for similar exchanges at the level of methionine residues in proteins. Moreover, oxidative refolding of the fully reduced [Sec(3),Sec(1 1),Nle(7)]-endothelin-1 fulfilled the expectation that the redox potential of the selenocysteines would dictate quantitative formation of the natural isomer. These results suggest that the selenocysteine approach, besides off ering an interesting chemical tool fur induction of correct oxidative foldi ng of multiple cysteine-containing peptides, should even allow for the prep aration of non-natural isomers and thus for studying conformational prefere nces of folding intermediates in peptides and proteins. (C) 1998 Academic P ress.