<i>In Vitro</i>Site-Specific Incorporation of Fluorescent Probes into β-Galactosidase

Abstract

Fluorescence spectroscopy is a powerful biophysical technique for studying protein structure, function, dynamics, and intermolecular interactions. Such studies are often conducted using intrinsic probes, such as tryptophan residues, or extrinsic probes introduced by post-translational modification, such as dansyl. Specificity, however, is often a concern since many proteins contain more than one tryptophan and chemical modification often will occur at more than one site. Herein we report the in vitro, site-specific incorporation of three fluorescent amino acid analogues, 5-hydroxytryptophan, 7-azatryptophan, and ε-dansyllysine, each of which was incorporated into β-galactosidase at a single designated site.