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Interactions of AsCy3 with cysteine-rich peptides.

Alexander SC, Schepartz A - Org. Lett. (2014)

Bottom Line: There is great interest in fluorogenic compounds that tag biomolecules within cells.Biarsenicals are fluorogenic compounds that become fluorescent upon binding four proximal Cys thiols, a tetracysteine (Cys(4)) motif.This work details interactions between the biarsenical AsCy3 and Cys(4) peptides.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and ‡Department of Molecular, Cellular and Developmental Biology, Yale University , New Haven, Connecticut 06520-8107, United States.

ABSTRACT
There is great interest in fluorogenic compounds that tag biomolecules within cells. Biarsenicals are fluorogenic compounds that become fluorescent upon binding four proximal Cys thiols, a tetracysteine (Cys(4)) motif. This work details interactions between the biarsenical AsCy3 and Cys(4) peptides. Maximal affinity was observed when two Cys-Cys pairs were separated by at least 8 amino acids; the highest affinity ligand bound in the nanomolar concentration range (K(app) = 43 nM) and with a significant (3.2-fold) fluorescence enhancement.

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Interactionsof AsCy3 with Cy3Tag and variants. (A) Sequence ofCy3Tag, TagΔ2, and TagΔ4 with Kapp values determined by FP. (B) Plot of the FI of 100 nM AsCy3 afterincubation with the Cy3Tag, TagΔ2, and TagΔ4. (C) Plotof the FP under identical conditions. Error bars show standard error.
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fig2: Interactionsof AsCy3 with Cy3Tag and variants. (A) Sequence ofCy3Tag, TagΔ2, and TagΔ4 with Kapp values determined by FP. (B) Plot of the FI of 100 nM AsCy3 afterincubation with the Cy3Tag, TagΔ2, and TagΔ4. (C) Plotof the FP under identical conditions. Error bars show standard error.

Mentions: To better explore the AsCy3·CyTag binding mode, we synthesizeda pair of CyTag variants in which one (TagΔ2) or both (TagΔ4)Cys-Cys motifs were replaced by Ala-Ala (Figure 2A). The interactions of TagΔ2 and TagΔ4 with AsCy3 wereevaluated by monitoring changes in both fluorescence intensity (Figure 2B) and fluorescence polarization (Figure 2C) as a function of peptide concentration. OnlyTagΔ2 showed evidence of an interaction with AsCy3 (Figure 2B). As was true for the CyTag interaction, the datacould be fit to a 1:1 binding isotherm, yielding a Kapp value of 960 ± 150 nM based on fluorescence intensitychanges and Kapp = 410 ± 92 nM basedon changes in fluorescence polarization. These Kapp values equal or exceed those determined for Cy3Tag itself,depending on the method (FI, 970 ± 140 nM; FP, 2.3 ± 0.6μM). The observation that AsCy3 interacts comparably with peptidescontaining one or two Cys-Cys motifs suggests that only one Cys-Cyspair in the Cy3Tag sequence contributes to complex stability.18 Indeed, the change in AsCy3 fluorescence emission(100 nM) in the presence of TagΔ2 (10 μM) is >60% oftheenhancement observed with CyTag. This observation indicates that thesecond Cys-Cys pair contributes minimally, if at all, to AsCy3 fluorogenicity,and the dye may only be partially bound to all four cysteines (Figure S1C in Supporting Information).


Interactions of AsCy3 with cysteine-rich peptides.

Alexander SC, Schepartz A - Org. Lett. (2014)

Interactionsof AsCy3 with Cy3Tag and variants. (A) Sequence ofCy3Tag, TagΔ2, and TagΔ4 with Kapp values determined by FP. (B) Plot of the FI of 100 nM AsCy3 afterincubation with the Cy3Tag, TagΔ2, and TagΔ4. (C) Plotof the FP under identical conditions. Error bars show standard error.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4334252&req=5

fig2: Interactionsof AsCy3 with Cy3Tag and variants. (A) Sequence ofCy3Tag, TagΔ2, and TagΔ4 with Kapp values determined by FP. (B) Plot of the FI of 100 nM AsCy3 afterincubation with the Cy3Tag, TagΔ2, and TagΔ4. (C) Plotof the FP under identical conditions. Error bars show standard error.
Mentions: To better explore the AsCy3·CyTag binding mode, we synthesizeda pair of CyTag variants in which one (TagΔ2) or both (TagΔ4)Cys-Cys motifs were replaced by Ala-Ala (Figure 2A). The interactions of TagΔ2 and TagΔ4 with AsCy3 wereevaluated by monitoring changes in both fluorescence intensity (Figure 2B) and fluorescence polarization (Figure 2C) as a function of peptide concentration. OnlyTagΔ2 showed evidence of an interaction with AsCy3 (Figure 2B). As was true for the CyTag interaction, the datacould be fit to a 1:1 binding isotherm, yielding a Kapp value of 960 ± 150 nM based on fluorescence intensitychanges and Kapp = 410 ± 92 nM basedon changes in fluorescence polarization. These Kapp values equal or exceed those determined for Cy3Tag itself,depending on the method (FI, 970 ± 140 nM; FP, 2.3 ± 0.6μM). The observation that AsCy3 interacts comparably with peptidescontaining one or two Cys-Cys motifs suggests that only one Cys-Cyspair in the Cy3Tag sequence contributes to complex stability.18 Indeed, the change in AsCy3 fluorescence emission(100 nM) in the presence of TagΔ2 (10 μM) is >60% oftheenhancement observed with CyTag. This observation indicates that thesecond Cys-Cys pair contributes minimally, if at all, to AsCy3 fluorogenicity,and the dye may only be partially bound to all four cysteines (Figure S1C in Supporting Information).

Bottom Line: There is great interest in fluorogenic compounds that tag biomolecules within cells.Biarsenicals are fluorogenic compounds that become fluorescent upon binding four proximal Cys thiols, a tetracysteine (Cys(4)) motif.This work details interactions between the biarsenical AsCy3 and Cys(4) peptides.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and ‡Department of Molecular, Cellular and Developmental Biology, Yale University , New Haven, Connecticut 06520-8107, United States.

ABSTRACT
There is great interest in fluorogenic compounds that tag biomolecules within cells. Biarsenicals are fluorogenic compounds that become fluorescent upon binding four proximal Cys thiols, a tetracysteine (Cys(4)) motif. This work details interactions between the biarsenical AsCy3 and Cys(4) peptides. Maximal affinity was observed when two Cys-Cys pairs were separated by at least 8 amino acids; the highest affinity ligand bound in the nanomolar concentration range (K(app) = 43 nM) and with a significant (3.2-fold) fluorescence enhancement.

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