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

Abstract

All known organisms encode 20 canonical amino acids by base triplets in the genetic code. The cellular translational machinery produces proteins consisting mainly of these amino acids. Several hundred natural amino acids serve important functions in metabolism, as scaffold molecules, and in signal transduction. New side chains are generated mainly by post-translational modifications, while others have altered backbones, such as the β- or γ-amino acids, or they undergo stereochemical inversion, e.g., in the case of D-amino acids. In addition, the number of non-canonical amino acids has further increased by chemical syntheses. Since many of these non-canonical amino acids confer resistance to proteolytic degradation, they are potential protease inhibitors and tools for specificity profiling studies in substrate optimization and enzyme inhibition. Other applications include in vitro and in vivo studies of enzyme kinetics, molecular interactions and bioimaging, to name a few. Amino acids with bio-orthogonal labels are particularly attractive, enabling various cross-link and click reactions for structure-functional studies. Here, we cover the latest developments in protease research with non-canonical amino acids, which opens up a great potential, e.g., for novel prodrugs activated by proteases or for other pharmaceutical compounds, some of which have already reached the clinical trial stage.

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

“…The general use of quadruplets seems therefore likely to be constrained, also because cells with a new genetic code containing 256 quadruplets would pose challenges in terms of metabolic burden and code degeneracy, as discussed recently. 299 OTS based on quadruplet codes or even the use of noncanonical base pairs could potentially be developed as an in vitro or in vivo platform which can operate as a subsystem (like a virtual machine in IT) for biopolymer biosynthesis. 300 For in vivo platforms, it is conceivable that engineered eukaryotic host cells could enable that by harnessing their compartmentalization capabilities.…”

“…However, special 5′ UTR had to be engineered for that making this a specific application. The general use of quadruplets seems therefore likely to be constrained, also because cells with a new genetic code containing 256 quadruplets would pose challenges in terms of metabolic burden and code degeneracy, as discussed recently . OTS based on quadruplet codes or even the use of noncanonical base pairs could potentially be developed as an in vitro or in vivo platform which can operate as a subsystem (like a virtual machine in IT) for biopolymer biosynthesis .…”

Noncanonical Amino Acids in Biocatalysis

“…Furthermore, mutation of key catalytic residues often dramatically reduces activity, which provides important but limited mechanistic insights. The incorporation of ncAAs can open new avenues to study complex biochemical mechanisms by allowing more nuanced perturbations to enzyme structure and function. ,,, Such modifications can be used to introduce spectroscopic probes, enable covalent trapping of intermediates and fine-tune active site environments by replacement of specific atoms or functional groups.…”

“…To date, these methods have been used to encode hundreds of structurally diverse ncAAs into proteins for diverse applications (Figure ). , Genetic code reprogramming has allowed introduction of new spectroscopic handles − and targeted replacement of individual atoms or functional groups, , providing new insights into how enzymes operate. These methods have also been used to develop engineered biocatalysts with augmented properties. − By combining genetic code reprogramming with directed evolution, it has also been possible to embed new modes of catalysis into proteins that would be difficult to access within the constraints of the genetic code. , …”

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

“…UAAs have found a wide range of utility in biological science. − However, in order for a UAA to be useful as a spectroscopic and/or imaging reporter of proteins, it must meet several requirements: (1) it should be able to produce a detectable, distinguishable, and interpretable signal (e.g., a vibrational or fluorescence signal) with desired spectroscopic properties; (2) it should be a simple derivative of one of the canonical amino acids; (3) it should be chemically stable; (4) it should not significantly perturb the structural and functional properties of the protein in question; and (5) it should be able to be incorporated into proteins via either a chemical or biological method.…”