Preparation and preliminary characterization of crystallizing fluorescent derivatives of chicken egg white lysozyme

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to the side chain carboxyl of Asp(101) using a carbodiimide coupling procedure. Asp(101) lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. Lucifer yellow and EDANS probes with iodoacetamide reactive groups have been bound to His(15), located on the "back side" of the molecule relative to the active site. All the derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching by added solutes. Quenching studies show that, as postulated, the Asp(101)-bound probes are partially sheltered from the bulk solution by their location within the active site cleft. Probes bound to His(15) have quenching constants about equal to those for the free probes, indicating that this site is highly exposed to the bulk solution. (C) 2001 Elsevier Science B.V. All rights reserved.