Latest methods of fluorescence-based protein crystal identification

Abstract

Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes. The introduction of fluorescence techniques has brought a powerful and versatile tool to the aid of the crystal grower. Trace fluorescent labeling, in which a fluorescent probe is covalently bound to a subpopulation (<0.5%) of the protein, enables the use of visible fluorescence. Alternatively, one can avoid covalent modification and use UV fluorescence, exploiting the intrinsic fluorescent amino acids present in most proteins. By the use of these techniques, crystals that had previously been obscured in the crystallization drop can readily be identified and distinguished from amorphous precipitate or salt crystals. Additionally, lead conditions that may not have been obvious as such under white-light illumination can be identified. In all cases review of the screening plate is considerably accelerated, as the eye can quickly note objects of increased intensity.

Multivalent Interactions by the Set8 Histone Methyltransferase With Its Nucleosome Substrate

“…To confirm the presence of Set8Δ1 in such crystals, we trace labeled Set8Δ1 with carboxyrhodamine [30–32], purified reconstituted Set8Δ1/RCC1/nucleosome complex by size exclusion chromatography, set up crystallization trials and examined resulting crystals with a fluorescence microscope (Supplementary Fig. 3a, b).…”

“…All proteins were analyzed by dynamic light scattering and determined to be non-aggregated. Selected Set8 proteins used for crystallization were N-terminally labeled with carboxyrhodamine as previously described [30–32]. Briefly, Set8 was reacted with two molar equivalents of 5-(and 6-)Carboxyrhodamine succinimidyl ester (Life Technologies) at pH 7.5 followed by purification using a Superdex 200 column.…”

Preparation, Crystallization, and Structure Determination of Chromatin Enzyme/Nucleosome Complexes

“…To short circuit this problem and streamline crystal screening, we use fluorescently labeled chromatin proteins during screening. This allows us to verify the presence of the chromatin protein in any crystallization hits in situ , following procedures developed more generally for protein crystallization (Meyer, Betzel, & Pusey, 2015; Pusey et al, 2015). …”

Improved protein-crystal identification by using 2,2,2-trichloroethanol as a fluorescence enhancer

“…Protein crystals typically fluoresce brightly when illuminated by the relevant excitation wavelengths, whereas salt crystals remain dark. In addition, owing to the denser protein packing in crystals compared with precipitate, protein crystals can be identified even if they are buried within precipitate and thus are barely visible under bright light conditions (Meyer et al, 2015). This enables the detection of protein crystals in a fully automated high-throughput setup (Forsythe et al, 2006).…”