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Trace fluorescent labeling for protein crystallization
Author(s) -
Pusey Marc,
Barcena Jorge,
Morris Michelle,
Singhal Anuj,
Yuan Qunying,
Ng Joseph
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x15008626
Subject(s) - fluorescence , protein crystallization , nucleation , crystallization , crystal (programming language) , fluorescent protein , quantum yield , chemistry , trace (psycholinguistics) , materials science , crystallography , photochemistry , analytical chemistry (journal) , optics , chromatography , green fluorescent protein , computer science , organic chemistry , physics , biochemistry , linguistics , philosophy , gene , programming language
Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace‐labeling approach, the protein is covalently derivatized with a high‐quantum‐yield visible‐wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate‐screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds `hidden' leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single‐pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV‐transparent.