In Vitro Site-Specific Incorporation of Fluorescent Probes into -Galactosidase

J. Am. Chem. Soc., 119 (1), 6 -11, 1997. 10.1021/ja963023f S0002-7863(96)03023-5

In Vitro Site-Specific Incorporation of Fluorescent Probes into -Galactosidase

Lance E. Steward, Cynthia S. Collins, Marcella A. Gilmore, Justin E. Carlson, J. B. Alexander Ross,* and A. Richard Chamberlin*

Contribution from the Department of Chemistry, University of California, Irvine, California 92697, and Department of Biochemistry, Mt. Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029

Received August 28, 1996

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 -dansyllysine, each of which was incorporated into -galactosidase at a single designated site.

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