RESUMO
Diversity in the biochemical workhorses of the cell-that is, proteins-is achieved by the innumerable permutations offered primarily by the 20 canonical L-amino acids prevalent in all biological systems. Yet, proteins are known to additionally undergo unusual modifications for specialized functions. Of the various post-translational modifications known to occur in proteins, the recently identified non-disulfide cross-links are unique, residue-specific covalent modifications that confer additional structural stability and unique functional characteristics to these biomolecules. We review an exclusive class of amino acid cross-links encompassing aromatic and sulfur-containing side chains, which not only confer superior biochemical characteristics to the protein but also possess additional spectroscopic features that can be exploited as novel chromophores. Studies of their in vivo reaction mechanism have facilitated their specialized in vitro applications in hydrogels and protein anchoring in monolayer chips. Furthering the discovery of unique canonical cross-links through new chemical, structural, and bioinformatics tools will catalyze the development of protein-specific hyperstable nanostructures, superfoods, and biotherapeutics.
RESUMO
Photo-actively modified natural amino acids have served as lucrative probes for precise mapping of the dynamics, interaction networks, and turnover of cytosolic proteins both inâ vivo and ex vivo. In our attempts to extend the utility of photoreactive reporters to map the molecular characteristics of vital membrane proteins, we carried out site-selective incorporation of 7-fluoro-indole in the human mitochondrial outer membrane protein VDAC2 (voltage-dependent anion channel isoform 2), with the aim of generating Trp-Phe/Tyr cross-links. Prolonged irradiation at 282â nm provided us with a surprisingly unusual fluorophore that displayed sizably red-shifted excitation (λex-max =280â nmâ360â nm) and emission (λem-max =330â nmâ430â nm) spectra that was reversible with organic solvents. By measuring the kinetics of the photo-activated cross-linking with a library of hVDAC2 variants, we demonstrate that formation of this unusual fluorophore is kinetically retarded, independent of tryptophan, and is site-specific. Using other membrane (Tom40 and Sam50) and cytosolic (MscR and DNA Pol I) proteins, we additionally show that formation of this fluorophore is protein-independent. Our findings reveal the photoradical-mediated accumulation of reversible tyrosine cross-links, with unusual fluorescent properties. Our findings have immediate applications in protein biochemistry and UV-mediated protein aggregation and cellular damage, opening avenues for formulating therapeutics that prolong cell viability in humans.