A Versatile Amino Acid Analogue of the Solvatochromic Fluorophore 4-NN-Dimethylamino-18-naphthalimide: A Powerful Tool for the Study of Dynamic Protein Interactions

摘要

We have developed a new unnatural amino acid based on the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) for application in the study of protein-protein interactions. The fluorescence quantum yield of this chromophore is highly sensitive to changes in the local solvent environment, demonstrating “switch-like” emission properties characteristic of the dimethylaminophthalimide family of fluorophores. In particular, this new species possesses a number of significant advantages over related fluorophores including greater chemical stability under a wide range of conditions, a longer wavelength of excitation (408 nm), and improved synthetic accessibility. This amino acid has been prepared as an Fmoc-protected building block and may readily be incorporated into peptides via standard solid-phase peptide synthesis (SPPS). A series of comparative studies are presented to demonstrate the advantageous properties of the 4-DMN amino acid relative to that of the previously reported 4-N,N-dimethylaminophthalimidoalanine (4-DAPA) and 6-N,N-dimethylamino-2,3-naphthalimidoalanine (6-DMNA) amino acids. Other commercially available solvatochromic fluorophores are also include in these studies. The potential of this new probe as a tool for the study of protein-protein interactions is demonstrated by introducing it into a peptide that is recognized by calcium-activated calmodulin. The binding interaction between these two components yields an increase in fluorescence emission greater than 900-fold.