Labeling Freedom for the Single Molecule Microscopist

Abstract

Observation of single molecule fluorescence has matured into a central tool to study biomolecular structure and dynamics. As hardware and data analysis technology dramatically improved over the last decade, site-specific labeling of proteins with small but highly photostable fluorescent dyes has turned into a major bottleneck for biological applications. In vitro, traditional approaches to label natural amino acids are the most widely applied, but due to the high abundance of even rare cysteines in larger biological machineries, few protein systems are accessible. Still more difficult to achieve is the ultimate goal of visualizing protein structure and dynamics in living cells and organisms. Compared to small organic fluorophores, however, fluorescent proteins, such as GFPs, are bigger and typically have worse photophysical properties, but since they can be genetically attached to any protein, they are the usual choice for in vivo studies. We have now developed a semi-synthetic strategy based on a novel artificial amino acid that is easily and site-specifically introduced into any protein by the natural machinery of the living cell. Expressed proteins only differ from their natural counterparts by very few atoms, constituting a ring-strained cyclooctyne functional group. We show that this completely inert and non-toxic group can be stably incorporated into any protein and readily reacts with commercially available single molecule fluorophores without the need of special reagents, catalyst or non-physiological buffer conditions. Similarly to fluorescent proteins, the dye attachment site is genetically encoded and will thus facilitate precise labeling of proteins in vivo by only changing a single amino acid. In fact, the speed and specificity of this method holds great promise for applications of single molecule and super resolution techniques in living cells, and new experimental results demonstrating this potential will be presented.