Proteins are the molecular machines of life, performing a myriad of functions inside every cell of our body. Proteins are assembled from small building blocks, the amino acids, by large protein factories called ribosomes. Understanding how proteins work is a quest of basic biology and medical research.
To study proteins inside of human cells, researchers have been using light microscopes for more than one hundred years. Thirty years ago, the cloning of the green fluorescent protein GFP, together with genetic engineering tools, revolutionized the field by enabling researchers to fuse a fluorescent ‘beacon’ to any protein of interest so that it can be directly observed in living cells using fluorescence microscopy. Fast forward, today’s microscopes achieve live imaging, at nanometer resolution, in multicolor, allowing researchers to resolve even the smallest subcellular structures and essentially watch protein at work.
Fluorescent proteins and other tools that are available to researchers have however one limitation: the size of the fluorescent tag is often equivalent to the size of a typical folded protein, thus adding a considerable molecular ‘cargo’ to the protein under study and potentially impacting its function. This can become a particular obstacle for the study of microproteins, a newly appreciated class of proteins that are much smaller than average. Such tiny proteins have often been overlooked in the past but seminal discoveries of microproteins with important biological functions have sparked growing interest by the research community.
We have made it a focus of our laboratory to tackle the challenges of discovering and characterizing microproteins. Here, we developed a method which allows fluorescent tagging of proteins with the smallest imaginable perturbation – a single amino acid – added genetically on either end of a (micro)protein of interest.
For the method, termed STELLA, a synthetic building block (a non-canonical “designer” amino acid, rather than one of the 21 canonical ones) is incorporated together with a larger tag using a technique termed genetic code expansion. The tag however is swiftly removed by the cell, leaving a single terminal designer amino acid on the protein of interest. As an advantage over existing labeling techniques relying on genetic code expansion, STELLA can thus be used to conveniently and universally label the termini of any proteins. While very similar to its natural counterpart, the designer amino acid introduces a peculiar chemical group into the protein that subsequently allows conjugation with a small organic fluorescent dye, now lighting up the protein of interest inside of the living cell.
Our research was funded by Karolinska Institutet SFO Molecular Biosciences, Ming Wai Lau Center for Reparative Medicine, Ragnar Söderbergs Stiftelse, Stiftelsen för Strategisk Forskning and Boehringer Ingelheim Fonds.