Abstract
We have designed and synthesized an azido-functionalized photoaffinity probe, [125I] GIF-0082, based on the structural modification of dantrolene. This compound specifically suppresses physiological Ca2+ release from sarcoplasmic reticulum (SR) of skeletal muscle without affecting Ca2+-induced Ca2+ release (CICR). Learning from the experience of handling the radioisotope (RI) labeled probe, we have developed a novel method of RI-free photoaffinity labeling in order to realize direct analysis of photolabeled proteins. This method utilizes a compact bifunctional probe consist of a photoreactive group and an aliphatic azido group that survives photolysis. By using this probe, a target protein is photo-cross-linked (Step 1), and then a detectable group is introduced by Staudinger-Bertozzi ligation using an alkyl azido moiety (Step 2). The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase using a diazido-functionalized cerivastatin derivative, photovastatin CAA 1, and a triarylphosphine derivative, GIF-0373, equipped with a fluorescein structure. The protocol has been also applied to the photolabeling of target proteins for a diazido-functionalized dantrolene derivative, GIF-0430.
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