NASA Logo

NTRS

NTRS - NASA Technical Reports Server

Back to Results
Preparation and Characterization of Fluorescent Derivatives of LysozymeFluorescence is one of the most versatile and powerful tools for the study of macromolecules. However, its use in macromolecular crystal growth studies is hampered by the necessity of preparing fluorescent derivatives where the probe does not markedly affect the crystal packing. Alternatively, one can prepare derivatives of limited utility if it is known that they will not affect the specific goals of a given study. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme, covalently attaching fluorescent probes to two different sites on the protein molecule. The first site is the side chain carboxyl group of ASP 101. Amine containing probes such as lucifer yellow, cascade blue, and 5- (2-aminoethyl) aminonapthalene-l-sulfonic acid (EDANS) have been attached 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. This is supported by the fact that all such derivatives have been found to crystallize, with the crystals being fluorescent. Tetragonal crystals of the lucifer yellow derivative have been found to diffract to at least 1.9 A resolution. X-ray diffraction data has been acquired and we are now working on the structure of this derivative. The second group of derivatives is to the N-terminal amine group. The derivatization reaction is performed by using a succinimidyl ester of the probe to be attached. Fluorescent probes such as pyrene acetic acid, 5-carboxyfluorescein, and Oregon green have been attached to this site. We have had little success in crystallizing these derivatives, probably because this site is part of the contact region between the 43 helix chains. However, these sites do not interfere with formation of the 43 helices and the derivatives are suitable for study of their formation in solution. The derivatives are being characterized by steady state and lifetime fluorescence methods, and the presentation will discuss these results.
Document ID
19990018628
Acquisition Source
Marshall Space Flight Center
Document Type
Reprint (Version printed in journal)
Authors
Smith, Lori
(Alabama Univ. Huntsville, AL United States)
Pusey, Marc
(NASA Marshall Space Flight Center Huntsville, AL United States)
Date Acquired
August 19, 2013
Publication Date
January 1, 1998
Subject Category
Chemistry And Materials (General)
Meeting Information
Meeting: Crystallization of Biological Macromolecules
Location: Granada
Country: Spain
Start Date: May 3, 1998
Distribution Limits
Public
Copyright
Work of the US Gov. Public Use Permitted.

Available Downloads

There are no available downloads for this record.
No Preview Available