Bind, catalyze, and quantify: a modern protein and enzyme engineering toolbox of genetically encoded non-canonical amino acids.

Non-canonical amino acids (ncAAs) are versatile molecular building blocks that can enhance nearly every aspect of protein engineering, from improving binding affinity to enabling precise quantitative analyses. This review highlights advances from 2020-2025 that demonstrate how expanding the amino acid repertoire unlocks new functionalities. We examine how genetically encoded ncAAs diversify and tune metal-binding properties, enabling programmable coordination and redox behavior in engineered enzymes. Further, we explore biocatalytic applications, including multi-fold activity enhancements in natural enzymes and the introduction of entirely novel reactivity in artificial systems. Finally, we discuss the growing use of ncAAs as intrinsic biophysical reporters, which support a wide range of spectroscopic methods for tracking structure, dynamics, and interactions at residue-level resolution. These capabilities establish ncAAs as essential tools that can be deployed at any stage of the protein design process, from constructing new catalytic centers to quantifying molecular behaviors in real time.