RUI: Development of Unnatural Tryptophan Derivatives to Expand Tryptophan Function and to Study Biological Catalysis
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Overview
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Unnatural amino acids (UAAs) have been developed to expand functionality of canonical amino acids for diverse applications including biophysical tags for protein function and dynamics, chemical handles for bioconjugation, thermodynamic probes for proton-coupled electron transfer (PCET), and photolabile UAAs. Of the 20 natural amino acids, tryptophan has the most chemically complex structure, equipped with an electron-rich ring that is amenable to oxidation and/or substitution. These properties have been exploited by Nature to mediate high energy PCET reactions in biological catalysis and as a building block for chemical modification in natural product biosynthesis. Despite this, there is a lack of tryptophan-based UAAs to study biological structure/catalysis and to expand Trp?s natural function. The overall goal of this project is to develop a series of novel, unnatural tryptophan derivatives that exhibit unique spectroscopic and physicochemical properties enabling interrogation of both PCET reactions and the role of protein structure/dynamics in biological catalysis. We have chosen 5-hydroxyindole (5-HOI) as the building block because it requires only two to three step synthetic routes from readily available precursors to generate a collection of halogenated derivatives with tunable acidities, reduction potentials and spectroscopic signatures. To enable site-specific incorporation into peptides and proteins, an evolved aminoacyl tRNA synthetase has been generated for 5-hydroxytryptophan (5-HOW), converted from 5-HOI using evolved tryptophan synthases. As a proof-of-concept, these new 5-HOW derivatives will be substituted for the single tryptophan in the model protein, azurin, in which the Trp has been shown to generate a stable radical, allowing us to test the impact of the protein environment on the physicochemical and spectroscopic parameters of 5-HOW and its oxidized sidechains.
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