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Title: Trace fluorescent labeling for protein crystallization

The presence of a covalently bound fluorescent probe at a concentration of <0.5% does not affect the outcome of macromolecule crystallization screening experiments. Additionally, the fluorescence can be used to determine new, not immediately apparent, lead crystallization conditions. 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, inmore » 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.« less
Authors:
; ; ; ; ;  [1]
  1. iXpressGenes Inc., 601 Genome Way, Huntsville, AL 35810 (United States)
Publication Date:
OSTI Identifier:
22389080
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta crystallographica. Section F, Structural biology communications; Journal Volume: 71; Journal Issue: Pt 7; Other Information: PMCID: PMC4498700; PMID: 26144224; PUBLISHER-ID: ic5095; PUBLISHER-ID: S2053230X15008626; OAI: oai:pubmedcentral.nih.gov:4498700; Copyright (c) Pusey et al. 2015; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; BYPASSES; COLOR; CRYSTALLIZATION; CRYSTALS; DIFFRACTION; FLUORESCENCE; ILLUMINANCE; MOLECULES; NUCLEATION; OPTIMIZATION; PLATES; PROBES; SCREENING; WAVELENGTHS