Genetically encoding phenyl azide chemistry: new uses and ideas for classical biochemistry

Abstract

Introducing new physicochemical properties into proteins through genetically encoded Uaa (unnatural amino acid) incorporation can lead to the generation of proteins with novel properties not normally accessible with the 20 natural amino acids. Phenyl azide chemistry represents one such useful addition to the protein repertoire. Classically used in biochemistry as a non-specific photochemical protein cross-linker, genetically encoding phenyl azide chemistry at selected residues provides more powerful routes to post-translationally modify protein function in situ. The two main routes are modulation by light (optogenetics) and site-specific bio-orthogonal modification (bioconjugation) via Click chemistry. In the present article, we discuss both approaches and their influence on protein function.

Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle

“…Protein modification using genetically encoded non-native chemistry has been used primarily for passive labelling with suitable reporter adducts (see 5,8,9 for recent reviews) rather than to modulate function. One versatile ncAA is azidophenylalanine (azF), as it allows incorporation of phenyl azide chemistry (a classic tool in biochemistry 10–12 ) into proteins. 13,14 Phenyl azide has two distinct chemistries that makes it particularly useful in terms of new PTM approaches: photochemical activation and bioorthogonal click chemistry (Fig.…”

“…18,25 SfGFP 26 is an intrinsically fluorescent protein derived from the original Aequorea victoria GFP. 27 Autofluorescent proteins such as GFP have proved to be an excellent model system for investigating and understanding the effect of ncAA incorporation on protein function (for examples see reviews 2,28 ); structural studies are crucial for understanding the molecular basis of action of ncAA engineering but are still relatively rare, 10,16,29–33 and even less so for ones that actively modulate function. Here we provide a timely structural insight and comparison concerning fluorescence switching on either click addition or irradiation of sfGFP containing the H148azF mutation (sfGFP 148azF ).…”

Photo‐Crosslinkable Unnatural Amino Acids Enable Facile Synthesis of Thermoresponsive Nano‐ to Microgels of Intrinsically Disordered Polypeptides

“…Using our previous work on controlling the size of ELP coacervates within aqueous microdroplets as a guideline, we designed two ELPs containing photo‐crosslinkable p AzF residues to serve as the constituent polypeptides for the gel particles ( Figure A; Table S1, Supporting Information). One polypeptide, termed photo‐crosslinkable ELP (PCE), is composed of 80 pentameric repeats of the VPGVG motif with four regularly spaced p AzF residues, a sufficient number to form a crosslinked hydrogel network .…”

Synthesis and Explosion Hazards of 4-Azido-l-phenylalanine

“…Irradiation of the phenyl azide with UV light furnishes a reactive nitrene species, which then causes structural rearrangement and crosslinking within the protein, resulting in altered fluorescence profiles. 11 Otting and co-workers have developed several multi-dentate ligands that are synthesized in situ from protein-incorporated 4 . 12 In complex with appropriate metal ions, the approach allows for site-specific introduction of EPR- and NMR-active labels that provide powerful, long-range structural information about their tethered proteins.…”