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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 Tag+n sequences. (A)Sequences of potential AsCy3 ligands and Kapp values determined by FP. (B) Plot illustrating relationship between Kapp and the number of amino acids separatingthe Cys-Cys motifs (n).
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fig3: Interactionsof AsCy3 with Tag+n sequences. (A)Sequences of potential AsCy3 ligands and Kapp values determined by FP. (B) Plot illustrating relationship between Kapp and the number of amino acids separatingthe Cys-Cys motifs (n).

Mentions: Next, we synthesized a secondset of potential AsCy3 ligands containingprogressively longer intervening sequences and evaluated their interactionswith AsCy3 using fluorescence intensity and polarization assays (Figure 3). These potential AsCy3 ligands contained from6 to 13 amino acids interposed between the two Cys-Cys motifs andwere largely unstructured at 30 μM in the absence of AsCy3,as judged by circular dichroism (CD) spectroscopy (5 mM phosphate(pH 7.5), 140 mM KCl, and 5 mM DTT) (Figure S3in Supporting Information). All of the second-generation peptidesevaluated formed complexes with AsCy3, exhibiting Kapp values between 49 nM and 1.3 μM in the absenceof EDT and BME. With one exception (Tag+2), the values determinedusing FI and FP agreed to within their 95% confidence intervals (Table S1 in Supporting Information). Notably,the fitted value of Kapp decreased asthe number of residues between the two Cys-Cys motifs increased from5 to 9, with the largest increase between Tag+3 and Tag+4 (Figure S4 in Supporting Information). The highestaffinity ligand was Tag+6, whose AsCy3 complex was characterized bya Kapp value of 94 ± 16 nM (FI); Kapp = 49 ± 13 nM by fluorescence polarization.Titration of AsCy3 (100 nM) and Tag+6 (30 uM) with between 5 nM and10 μM EDT led to a systematic decrease in fluorescence emissionat 580 nm. This decrease could be fit to yield an inhibition constant(Ki) of 9.3 ± 4.6 μM (Figure S5 in Supporting Information), in agreementwith the value determined on the basis of competition with CyTag (Ki = 5.6 ± 0.9 μM). Thus, Tag+6 bindsAsCy3 more than 100 times more favorably than EDT or CyTag.


Interactions of AsCy3 with cysteine-rich peptides.

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

Interactionsof AsCy3 with Tag+n sequences. (A)Sequences of potential AsCy3 ligands and Kapp values determined by FP. (B) Plot illustrating relationship between Kapp and the number of amino acids separatingthe Cys-Cys motifs (n).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Interactionsof AsCy3 with Tag+n sequences. (A)Sequences of potential AsCy3 ligands and Kapp values determined by FP. (B) Plot illustrating relationship between Kapp and the number of amino acids separatingthe Cys-Cys motifs (n).
Mentions: Next, we synthesized a secondset of potential AsCy3 ligands containingprogressively longer intervening sequences and evaluated their interactionswith AsCy3 using fluorescence intensity and polarization assays (Figure 3). These potential AsCy3 ligands contained from6 to 13 amino acids interposed between the two Cys-Cys motifs andwere largely unstructured at 30 μM in the absence of AsCy3,as judged by circular dichroism (CD) spectroscopy (5 mM phosphate(pH 7.5), 140 mM KCl, and 5 mM DTT) (Figure S3in Supporting Information). All of the second-generation peptidesevaluated formed complexes with AsCy3, exhibiting Kapp values between 49 nM and 1.3 μM in the absenceof EDT and BME. With one exception (Tag+2), the values determinedusing FI and FP agreed to within their 95% confidence intervals (Table S1 in Supporting Information). Notably,the fitted value of Kapp decreased asthe number of residues between the two Cys-Cys motifs increased from5 to 9, with the largest increase between Tag+3 and Tag+4 (Figure S4 in Supporting Information). The highestaffinity ligand was Tag+6, whose AsCy3 complex was characterized bya Kapp value of 94 ± 16 nM (FI); Kapp = 49 ± 13 nM by fluorescence polarization.Titration of AsCy3 (100 nM) and Tag+6 (30 uM) with between 5 nM and10 μM EDT led to a systematic decrease in fluorescence emissionat 580 nm. This decrease could be fit to yield an inhibition constant(Ki) of 9.3 ± 4.6 μM (Figure S5 in Supporting Information), in agreementwith the value determined on the basis of competition with CyTag (Ki = 5.6 ± 0.9 μM). Thus, Tag+6 bindsAsCy3 more than 100 times more favorably than EDT or CyTag.

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.

Show MeSH