Minimalistic probes for protein folding and structure studies

Site-specific incorporation of fluorescent probes into proteins enhances the ability of researchers to study protein folding and structural dynamics. Currently, the most common fluorescent probe for proteins is green fluorescent protein (GFP). However, the use of GFP, a 28 kDa protein, requires its covalent attachment to a protein of interest. Additionally, this attachment is restricted to either the N- or C-terminus of the protein, drastically limiting the scope of experiments possible. Alternative technologies for the addition of novel fluorescence properties to proteins exist, however they are also limited by the probe size or its method of attachment. Since many proteins of interest are less than or equal to the size of these fluorescent auxiliaries, it is likely that the covalent fusion of the fluorescent apparatus will significantly perturb the native fold or function of the molecule of interest. As such, the development of minimalistic probes for protein functional studies is of great interest to the biochemical community. Acridon-2-ylalanine (Acd) is a minimalistic fluorescent unnatural amino acid (flUAA) that possess a long lifetime, resistance to photobleaching, a near unity quantum yield, and visible wavelength emissions. While there existed literature precedent for the synthesis of Acd, these methods required harsh conditions and reagents that precluded a high-yielding, scalable synthesis of the flUAA. To this end, we have devised an efficient, scalable 5-step synthesis utilizing Tyr that yields Acd in 87 % overall yield. Recently, we have shown that Acd can be incorporated into proteins in vivo and can be a valuable probe of protein-peptide interactions and protein conformational change due to its blue-wavelength excitation, unique solvatochromic properties, and ability to participate in energy transfer with endogenous amino acids (Trp and Tyr), exogenous fluorophores (methoxycoumarin), and the lanthanide ion Eu 3+. Furthermore, as the need for minimalistic probes is not limited to fluorescence applications, we have collaborated with the Deutsch laboratory to develop methods for the synthesis of unnatural amino acids capable of probing the interaction between the ribosomal exit tunnel and nascent peptides