Interactions of AsCy3 with cysteine-rich peptides

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.

Figure 1

AsCy3 and its interactions with Cy3Tag. (A) Sequence of Cy3Tag (left) and structure of AsCy3 (right). (B) Plots illustrating the changes in FI (left) and FP (right) of 0.1 μM AsCy3 after incubation with the [Cy3Tag] indicated in the presence (closed) and absence (open) of 100 μM EDT and 1 mM BME. (C) Competition between 10 μM CyTag and [EDT] for 0.1 μM AsCy3, measured by changes in FI. Errors show standard error. (D) Minimized structure (Gaussian) of the hypothetical complex between AsCy3 and Cy3Tag.

Figure 2

Interactions of AsCy3 with Cy3Tag and variants. (A) Sequence of Cy3Tag, TagΔ2, and TagΔ4 with K_app values determined by FP. (B) Plot of the FI of 100 nM AsCy3 after incubation with the Cy3Tag, TagΔ2, and TagΔ4. (C) Plot of the FP under identical conditions. Error bars show standard error.

Figure 3

Interactions of AsCy3 with Tag+n sequences. (A) Sequences of potential AsCy3 ligands and K_app values determined by FP. (B) Plot illustrating relationship between K_app and the number of amino acids separating the Cys-Cys motifs (n).

Figure 4

Interactions of AsCy3 with Tag+6 and Tag+6Δ2. (A) Sequences of Tag+6 and Tag+6Δ2 with K_app values measured by FP. (B) Plot of the FI of 100 nM AsCy3 after incubation with the indicated [Tag+6] and [Tag+6Δ2]. (C) Plot of the FP under identical conditions. Error bars represent the standard error.