Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1-47 of hirudin as a probe of hirudin folding and thrombin recognition

摘要: 7-Azatryptophan (AW), a noncoded isostere of tryptophan (W), possesses interesting spectral properties. In particular, the presence nitrogen atom at position 7 in indolyl nucleus AW results red shift absorption maximum and fluorescence emission by 10 46 nm, respectively, compared to W. present work, we report chemical synthesis conformational functional characterization an analog (denoted as Y3AW) N-terminal domain 1–47 hirudin, highly potent thrombin inhibitor, which Tyr 3 has been replaced AW. The obtained were with those cooresponding Y3W analog. We found that replacement W → reduces affinity for 10-fold, likely because lower hydrophobicity Measurements resonance energy transfer effect, was observed between Tyr13 amino acid upon disulfide-coupled folding, demonstrate behaves better acceptor than studying protein renaturation. interaction Y3AW studied exciting sample 320 nm recording change on binding enzyme. Our indicate hirudin Y3AW–thrombin complex is strongly quenched, possibly two structural water molecules hirudin–thrombin interface can promote nonradiative decay excited state. data herein reported incorporation be broad applicability study folding protein–protein interaction.