Trace fluorescent labeling for high-throughput crystallography

Abstract

Covalent labeling of macromolecules with trace levels (<1%) of a fluorescent dye is proposed as a means to facilitate finding or detecting crystals in crystallization drops. To test the effects of labeled protein concentration on the resulting X-ray diffraction data, experiments were carried out with the model proteins insulin, ribonuclease, lysozyme and thaumatin, which were labeled with the fluorescent dye carboxyrhodamine. All proteins were labeled on their N-terminal amine and lysozyme was also labeled randomly on lysine side chains in a separate series of experiments. Ribonuclease and N-terminal amine-labeled lysozyme crystals were poorly formed at 10% label concentration and these were not used in subsequent diffraction experiments. All model proteins were tested to 5% labeled protein, and thaumatin and randomly labeled lysozyme gave well formed crystals to 10% labeled protein. In all cases tested, the presence of the label was found to not significantly affect the X-ray diffraction data quality obtained. Qualitative visual-inspection experiments over a range of label concentrations indicated that optimum derivatization levels ranged from 0.025-0.05% for insulin to 0.1-0.25% for thaumatin. Light intensity is a simpler search parameter than straight lines and by virtue of being the most densely packed phase, labeled crystals should be the most intense light sources under fluorescent illumination. For both visual and automated methods of crystal detection, label intensity is a simpler and potentially more powerful search parameter. Screening experiments using the proteins canavalin, beta-lactoglobulins A and B and chymotrypsinogen, all at 0.5% label concentration, demonstrated the utility of this approach to rapidly finding crystals, even when obscured by precipitate. The use of trace-labeled protein is also proposed to be useful for the automated centering of crystals in X-ray beamlines.

Introduction to protein crystallization

“…Fluorescence methods may be based on the intrinsic fluorescence of proteins containing aromatic amino acids and nucleic acids composed of nucleotides. Alternately, the methods may require that some small fraction of the macromolecule be labeled with a fluorescent dye (Forsythe et al, 2006). Somewhat surprisingly, the labels appear not to interfere with crystallization, and this methods holds considerable promise.…”

Optimizing Associative Experimental Design for Protein Crystallization Screening

“…Plate results are scored over the range 0 to 9, as listed in Table I. Since we are using trace fluorescent labeling ( TFL ) [19], a score of 4 is assigned to outcomes giving “bright spot” lead conditions. For AED let…”

“…The previous TFL experiments on a range of proteins showed no effect on the protein crystallization process or the X-ray diffraction results [18], [19]. As the method is used to identify crystals and likely crystallization conditions, if there is any concern then once these have been found subsequent crystals for diffraction analysis can be grown using protein which has not been labeled.…”

Crystallizing Membrane Proteins for Structure-Function Studies Using Lipidic Mesophases

“…Detection by UV fluorescence is particularly powerful and convenient for tryptophancontaining proteins. Fluorescence labelling [48] is also a route worth considering for the sensitive detection of early hits. SONICC (second-order non-linear optical imaging of chiral crystals) is a novel approach introduced by G. Simpson.…”