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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.

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Fluorescence spectra in the context of the applied excitation spectra of the four aromatic amino acids tryptophan (a), tyrosine (b), phenylalanine (c) and histidine (d) in water at 1.8 mM. The dashed line indicates an excitation wavelength of 300 nm. For intrinsic fluorescence only tryptophan is relevant. Tyrosine fluorescence excitation is irrelevant for intrinsic fluorescence imaging owing to its weak absorption when illuminated with the applied illumination spectrum.
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fig2: Fluorescence spectra in the context of the applied excitation spectra of the four aromatic amino acids tryptophan (a), tyrosine (b), phenylalanine (c) and histidine (d) in water at 1.8 mM. The dashed line indicates an excitation wavelength of 300 nm. For intrinsic fluorescence only tryptophan is relevant. Tyrosine fluorescence excitation is irrelevant for intrinsic fluorescence imaging owing to its weak absorption when illuminated with the applied illumination spectrum.

Mentions: It has long been known that proteins contain four aromatic amino-acid residues (tryptophan, tyrosine, phenylalanine and histidine) which may contribute to the intrinsic fluorescence of a protein (Asanov et al., 2001 ▶). In contrast to fluorescence imaging of proteins based on trace fluorescence labeling, intrinsic fluorescence imaging rests predominantly on the fluorescence properties of tryptophan. Tryptophan has a fluorescence excitation maximum at a wavelength of 280 nm with maximum emitted fluorescence light at 350 nm (Teale & Weber, 1957 ▶; Fig. 2 ▶a) depending on the polarity of its close environment. Most plate-covering materials have a low opacity for UV wavelengths, especially the glass used for cover slips, glass cover plates and capillaries. An effective excitation spectrum for intrinsic fluorescence imaging as a detection tool is therefore expected to be in the range of 280 nm to approximately 300 nm, owing to the emission spectra of the available UV-light sources. Interestingly, for intrinsic fluorescence imaging of protein crystals, another part of the spectrum has been determined to be very useful for protein crystals enclosed in crystallization containers (Dierks et al., 2010 ▶).


Latest methods of fluorescence-based protein crystal identification.

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

Fluorescence spectra in the context of the applied excitation spectra of the four aromatic amino acids tryptophan (a), tyrosine (b), phenylalanine (c) and histidine (d) in water at 1.8 mM. The dashed line indicates an excitation wavelength of 300 nm. For intrinsic fluorescence only tryptophan is relevant. Tyrosine fluorescence excitation is irrelevant for intrinsic fluorescence imaging owing to its weak absorption when illuminated with the applied illumination spectrum.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Fluorescence spectra in the context of the applied excitation spectra of the four aromatic amino acids tryptophan (a), tyrosine (b), phenylalanine (c) and histidine (d) in water at 1.8 mM. The dashed line indicates an excitation wavelength of 300 nm. For intrinsic fluorescence only tryptophan is relevant. Tyrosine fluorescence excitation is irrelevant for intrinsic fluorescence imaging owing to its weak absorption when illuminated with the applied illumination spectrum.
Mentions: It has long been known that proteins contain four aromatic amino-acid residues (tryptophan, tyrosine, phenylalanine and histidine) which may contribute to the intrinsic fluorescence of a protein (Asanov et al., 2001 ▶). In contrast to fluorescence imaging of proteins based on trace fluorescence labeling, intrinsic fluorescence imaging rests predominantly on the fluorescence properties of tryptophan. Tryptophan has a fluorescence excitation maximum at a wavelength of 280 nm with maximum emitted fluorescence light at 350 nm (Teale & Weber, 1957 ▶; Fig. 2 ▶a) depending on the polarity of its close environment. Most plate-covering materials have a low opacity for UV wavelengths, especially the glass used for cover slips, glass cover plates and capillaries. An effective excitation spectrum for intrinsic fluorescence imaging as a detection tool is therefore expected to be in the range of 280 nm to approximately 300 nm, owing to the emission spectra of the available UV-light sources. Interestingly, for intrinsic fluorescence imaging of protein crystals, another part of the spectrum has been determined to be very useful for protein crystals enclosed in crystallization containers (Dierks et al., 2010 ▶).

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