Limits...
Latest methods of fluorescence-based protein crystal identification.

Meyer A, Betzel C, Pusey M - Acta Crystallogr F Struct Biol Commun (2015)

Bottom Line: Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes.Alternatively, one can avoid covalent modification and use UV fluorescence, exploiting the intrinsic fluorescent amino acids present in most proteins.In all cases review of the screening plate is considerably accelerated, as the eye can quickly note objects of increased intensity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, c/o DESY Building 22a, Notkestrasse 85, 22607 Hamburg, Germany.

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.

Show MeSH

Related in: MedlinePlus

(a, b) Hen egg-white lysozyme crystals among sodium chloride crystals illuminated with filtered radiation from a mercury vapor lamp (a) and white light (b). The protein crystals show the typical intrinsic blue fluorescence of tryptophan, while the salt crystals are invisible when excited with this spectrum. (c) Crystals of the 40-mer RNA l-oligonucleotide in complex with the protein l-CCP2 illuminated with bright light. (d) The same crystals when illuminated with the excitation spectrum; crystals of the protein–RNA complex crystals show the intrinsic blue fluorescence of tryptophan. (e) The same crystals after the addition of SYBR Gold fluorescence stain (∼1:1000); the crystals show characteristic green fluorescence, which would not be the case if nucleic acids were not present in the crystals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4321462&req=5

fig5: (a, b) Hen egg-white lysozyme crystals among sodium chloride crystals illuminated with filtered radiation from a mercury vapor lamp (a) and white light (b). The protein crystals show the typical intrinsic blue fluorescence of tryptophan, while the salt crystals are invisible when excited with this spectrum. (c) Crystals of the 40-mer RNA l-oligonucleotide in complex with the protein l-CCP2 illuminated with bright light. (d) The same crystals when illuminated with the excitation spectrum; crystals of the protein–RNA complex crystals show the intrinsic blue fluorescence of tryptophan. (e) The same crystals after the addition of SYBR Gold fluorescence stain (∼1:1000); the crystals show characteristic green fluorescence, which would not be the case if nucleic acids were not present in the crystals.

Mentions: An identical excitation spectrum has been tested using Crystal-Tube Gel Tube R glass capillaries (GT-R; CFS-MB2004-CRT200). These have a thick wall of approximately 0.5 mm (Figs. 4 ▶a and 4 ▶b). GT-R were used to apply a variation of the counter-diffusion liquid-to-liquid crystallization approach. The capillary was filled with protein solution and a piece of gel tubing was attached to one end of the capillary (McPherson, 1999 ▶). For intrinsic fluorescence imaging, the capillaries need to be taken out of the Granada Crystallization boxes owing to the intrinsic fluorescence properties of the box itself. Crystals of mistletoe lectin I were illuminated with white light and the excitation spectra were recorded (Figs. 4 ▶ ▶a and 4 ▶b, respectively). Additionally, common glass capillaries with thin walls of approximately 0.1 mm have been investigated (Figs. 4 ▶c and 4 ▶d). With the assumption that most precipitants contain no aromatic groups, intrinsic fluorescence can distinguish between protein and precipitant crystals. Fig. 5 ▶ shows lysozyme crystals among sodium chloride crystals in the well of an MRC2 crystallization plate (MRC 96-well crystallization plate; catalog No. MD11-00U-100, Molecular Dimensions, UK), covered with sealing film (ClearVue Sheets; catalog No. MD6-01S) when illuminated with the excitation spectrum (Fig. 5 ▶a) and when illuminated with white light (Fig. 5 ▶b).


Latest methods of fluorescence-based protein crystal identification.

Meyer A, Betzel C, Pusey M - Acta Crystallogr F Struct Biol Commun (2015)

(a, b) Hen egg-white lysozyme crystals among sodium chloride crystals illuminated with filtered radiation from a mercury vapor lamp (a) and white light (b). The protein crystals show the typical intrinsic blue fluorescence of tryptophan, while the salt crystals are invisible when excited with this spectrum. (c) Crystals of the 40-mer RNA l-oligonucleotide in complex with the protein l-CCP2 illuminated with bright light. (d) The same crystals when illuminated with the excitation spectrum; crystals of the protein–RNA complex crystals show the intrinsic blue fluorescence of tryptophan. (e) The same crystals after the addition of SYBR Gold fluorescence stain (∼1:1000); the crystals show characteristic green fluorescence, which would not be the case if nucleic acids were not present in the crystals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321462&req=5

fig5: (a, b) Hen egg-white lysozyme crystals among sodium chloride crystals illuminated with filtered radiation from a mercury vapor lamp (a) and white light (b). The protein crystals show the typical intrinsic blue fluorescence of tryptophan, while the salt crystals are invisible when excited with this spectrum. (c) Crystals of the 40-mer RNA l-oligonucleotide in complex with the protein l-CCP2 illuminated with bright light. (d) The same crystals when illuminated with the excitation spectrum; crystals of the protein–RNA complex crystals show the intrinsic blue fluorescence of tryptophan. (e) The same crystals after the addition of SYBR Gold fluorescence stain (∼1:1000); the crystals show characteristic green fluorescence, which would not be the case if nucleic acids were not present in the crystals.
Mentions: An identical excitation spectrum has been tested using Crystal-Tube Gel Tube R glass capillaries (GT-R; CFS-MB2004-CRT200). These have a thick wall of approximately 0.5 mm (Figs. 4 ▶a and 4 ▶b). GT-R were used to apply a variation of the counter-diffusion liquid-to-liquid crystallization approach. The capillary was filled with protein solution and a piece of gel tubing was attached to one end of the capillary (McPherson, 1999 ▶). For intrinsic fluorescence imaging, the capillaries need to be taken out of the Granada Crystallization boxes owing to the intrinsic fluorescence properties of the box itself. Crystals of mistletoe lectin I were illuminated with white light and the excitation spectra were recorded (Figs. 4 ▶ ▶a and 4 ▶b, respectively). Additionally, common glass capillaries with thin walls of approximately 0.1 mm have been investigated (Figs. 4 ▶c and 4 ▶d). With the assumption that most precipitants contain no aromatic groups, intrinsic fluorescence can distinguish between protein and precipitant crystals. Fig. 5 ▶ shows lysozyme crystals among sodium chloride crystals in the well of an MRC2 crystallization plate (MRC 96-well crystallization plate; catalog No. MD11-00U-100, Molecular Dimensions, UK), covered with sealing film (ClearVue Sheets; catalog No. MD6-01S) when illuminated with the excitation spectrum (Fig. 5 ▶a) and when illuminated with white light (Fig. 5 ▶b).

Bottom Line: Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes.Alternatively, one can avoid covalent modification and use UV fluorescence, exploiting the intrinsic fluorescent amino acids present in most proteins.In all cases review of the screening plate is considerably accelerated, as the eye can quickly note objects of increased intensity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, c/o DESY Building 22a, Notkestrasse 85, 22607 Hamburg, Germany.

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.

Show MeSH
Related in: MedlinePlus