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A Bifunctional Noncanonical Amino Acid: Synthesis, Expression, and Residue-Specific Proteome-wide Incorporation
journal contribution
posted on 2018-06-22, 20:13 authored by Jan-Erik Hoffmann, Dmytro Dziuba, Frank Stein, Carsten SchultzMapping
of weak and hence transient interactions between low-abundance
interacting molecules is still a major challenge in systems biology
and protein biochemistry. Therefore, additional system-wide acting
tools are needed to determine protein interactomics. Most important
are reagents that can be applied at any kind of protein interface
and the possibility to enrich cross-linked fragments with high efficiency.
In this study, we report the synthesis of a novel noncanonical amino
acid that features a diazirine group for ultraviolet cross-linking
as well as an alkyne group for labeling by click chemistry. This bifunctional
amino acid, called PrDiAzK, may be inserted into almost any protein
interface with minimal structural perturbation using genetic code
expansion. We demonstrate that PrDiAzK can be site-selectively incorporated
into proteins in both bacterial and mammalian cell cultures, and we
show that PrDiAzK allows protein labeling as well as cross-linking.
In addition, we tested PrDiAzK for proteome-wide incorporation via
stochastic orthogonal recoding of translation, implying potential
applications in system-wide mapping of protein–protein interactions
in the future.