Directed Self-Assembly of Trimeric DNA-Bindingchiral Miniprotein Helicates

Abstract

We propose that peptides are highly versatile platforms for the precise design of supramolecular metal architectures, and particularly, for the controlled assembly of helicates. In this context, we show that the bacteriophage T4 Fibritin foldon (T4Ff) can been engineered on its N-terminus with metal-chelating 2,2'-bipyridine units that stereoselectively assemble in the presence of Fe(II) into parallel, three-stranded peptide helicates with preferred helical orientation. Modeling studies support the proposed self-assembly and the stability of the final helicate. Furthermore, we show that these designed mini-metalloproteins selectively recognize three-way DNA junctions over double-stranded DNA.

Keywords: DNA recognition; coordination chemistry; enantioselectivity; metallopeptide; peptide motifs; self-assembly water.

Figure 1

(A) Structural elements and sequence of the natural T4Ff, and proposed structure of the (βAlaBpy)₂-T4Ff helicate at the N-terminus of the T4Ff scaffold. The three chains of the T4Ff are shown with different colors (orange, blue, light gray) for clarity. The ΛΛ- chirality is induced by the natural twisting of the T4Ff N-terminal polyproline helices. (B) Synthetic procedure for obtaining the T4Ff helicates, and structure of the chelating Fmoc-βAlaBpy-OH amino acid.

Figure 2

Fluorescence titration of a 3 μM (9 μM monomer) solution of [(βAlaBpy)₂-T4Ff]₃ with increasing concentrations of Fe(II). Inset shows emission at 420 nm upon excitation at 305 nm with increasing concentrations of Fe(II), and the best fit to a 1:2 binding mode (Hellman and Fried, ; Peberdy et al., 2007). Experiments were made in triplicate. Right. Circular Dichroism of a 6 μM solution (18 μM monomer) of [(βAlaBpy)₂-T4Ff]₃ (dashed line) and in the presence of 90 μM Fe(II) (solid line). All experiments were made in 1 mM phosphate buffer, pH 6.5, 10 mM NaCl at 20°C.

Figure 3

First cluster representative frame of the MD trajectory for the ΛΛ-[(βAlaBpy)₂-T4Ff]₃Fe${}_2^{+4}$ system showing the stable structure of the T4Ff domain. Note the flexible hinge region between the rigid helicate and the T4Ff domain.

Figure 4

(Left) Anisotropy titration of [(βAlaBpy)₂-T4Ff]₃Fe₂ in 1 mM phosphate buffer, 10 mM NaCl with increasing concentrations of tw-DNA. The best fit to a 1:1 binding mode is shown (curve fitting was performed using DynaFit).(Kuzmic, 1996, 2009) tw-DNA sequences: 5′–CAC CGC TCT GGT CCT C−3′; 5′–CAG GCT GTG AGC GGT G−3′; 5′–GAG GAC CAA CAG CCT G−3′. Right: Model of the interaction between the [(βAlaBpy)₂-T4Ff]₃Fe₂ and the three-way junction, based on the reported pdb structures of an helicate bound to a three-way junction (pdb code 4NCU), and the structure of the fibritin foldon (pdb code 2ET0; Oleksy et al., 2006).

Figure 5

EMSA DNA binding studies results for [(βAlaBpy)₂-T4Ff]₃Fe₂ helicate. Lanes 1–6, 200 nM tw-Rho-DNA with 0, 150, 250, 500, 1,000, and 2,000 nM of [(βAlaBpy)₂-T4Ff]₃ and 14 eq. of (NH4)₂Fe(SO₄)₂ ∙ 6 H₂O in each lane; lanes 7–10, 200 nM dsDNA with 0, 500, 1,000, and 2,000 nM of [(βAlaBpy)₂-T4Ff]₃ and 14 eq. of (NH4)₂Fe(SO₄)₂ ∙ 6 H₂O in each lane. Samples were resolved on a 10% nondenaturing polyacrylamide gel and 1 × TBE buffer over 35 min at 25°C, and stained with SyBrGold (5 μL in 50 mL of 0.5 × TBE) for 10 min, followed by fluorescence visualization. Oligonucleotide sequences: tw-DNA, 5′–CAC CGC TCT GGT CCT C−3′; 5′–CAG GCT GTG AGC GGT G−3′; 5′–GAG GAC CAA CAG CCT G−3′; dsDNA (only one strand shown) 5′–AAC ACA TGC AGG ACG GCG CTT−3′.