Genetically encoded crosslinkers to address protein–protein interactions

Abstract

Noncanonical amino acids (ncAAs) for photo‐ and chemical crosslinking are powerful biochemical tools for studying and manipulating interactions between proteins both in vitro and in intact cells. Since the first crosslinking ncAAs were genetically encoded about 20 years ago, the technology has now ripened beyond the proof‐of‐principle demonstrations and is contributing to the study of relevant biological questions in the frame of modern integrative approaches. Here, we provide an overview of available photo‐activatable ncAAs for photo‐crosslinking and electrophilic ncAAs for genetically encoded chemical crosslinking (GECX), with a major focus on the most recent entries such as ncAAs for SuFEx click chemistry and photo‐activatable ncAAs for chemical crosslinking. We present recent examples of the application of genetically encoded crosslinkers to capture protein–protein interactions and identify interaction partners in live cells, to investigate molecular mechanisms of protein function, to stabilize protein complexes for structural studies, to derive structural information about protein complexes from the physiological cell environment, up to perspective applications of GECX‐ncAAs for the development of covalent drugs.

Evolution of Pyrrolysyl-tRNA Synthetase: From Methanogenesis to Genetic Code Expansion

“…The ability to make proteins containing new-to-nature noncanonical amino acids (ncAAs) has vastly expanded the chemical and functional space of proteins. This expansion has led to a huge increase in applications in basic and applied sciences, including medical therapies. − The specific incorporation of ncAAs relies on orthogonal translation systems (OTSs) consisting of engineered orthogonal aminoacyl-tRNA synthetase/tRNA pairs. The most common site-specific incorporation method is amber stop codon suppression (SCS).…”

“…Post-translational modifications (blue), ncAAs containing bioorthogonal groups and are possible targets for site-specific bioconjugation (red), photocaged ncAAs or cAAs (magenta), ncAAs containing functional groups which are useful for spectroscopic applications (orange), photo-or proximity triggered crosslinkable ncAAs (violet), fluorescent ncAAs (cyan). groups, 160,161 PTMs, 17,162 cross-linker, 11 elucidating enzyme substrates by trapping, 21 photo-and chemically caged amino acids, 163−167 and also combinations of these functions, e.g., photo controlled bioconjugation/cross-linking/trapping. 168−170 At a cellular level, GCE can be used to elucidate protein localization and interactions using methods like microscopy 132,171−173 or mass-spectroscopy-based proteomics.…”

Non-Canonical Amino Acids in Analyses of Protease Structure and Function

“…Site-specific cross-linking is a highly desired approach to investigating protein interactions in structural proteomics and in living cells [ 354 , 355 ]. For more in-depth details on this topic, see the highly topical review of Aydin and Conin, since our focus is on examples of proteases [ 356 ]. Thus, keratinase KerBL (subtilisin Carlsberg, S08.001) from Bacillus licheniformis was internally cross-linked by the reaction of the uAAs bromo-propyl/butyl-Tyr with Cys mutants, which stabilized flexible regions [ 357 ].…”

Covalent crosslinking in gas-phase biomolecular ions. An account and perspective

“…1), are recognized by the transfer RNAs for the corresponding standard amino acids and can be incorporated into expressed proteins to be used for carbene-based photo crosslinking. 37 An excellent recent review of crosslinking in solution combined with mass spectrometry analysis has been published by Sinz and coworkers. 1…”