Isomorphous replacement of cystine with selenocystine in endothelin: oxidative refolding, biological and conformational properties of [Sec3,Sec11,Nle7]-endothelin-1.

Air re-oxidation of fully reduced human endothelin-1 under optimized conditions 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 the recently determined highly reducing redox potential of selenocysteine (-381 mV) in peptides, the half-cystine residues Cys3 and Cys11 of the natural isomer of endothelin-1 were replaced by selenocysteine. Taking advantage of the high stability of the diselenide group toward reducing agents for disulfides a regioselective disulfide bridging of the second cysteine pair allowed for straightforward preparation of the [Sec3,Sec11, Nle7]-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 residues with selenocysteine was without detectable effect on receptor recognition and signal transduction. Both findings strongly support that the exchange of sulfur against selenium produces a fully isomorphous molecule as recently observed for similar exchanges at the level of methionine residues in proteins. Moreover, oxidative refolding of the fully reduced [Sec3,Sec11,Nle7]-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 offering an interesting chemical tool for induction of correct oxidative folding of multiple cysteine-containing peptides, should even allow for the preparation of non-natural isomers and thus for studying conformational preferences of folding intermediates in peptides and proteins. Copyright 1998 Academic Press