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. 2014 Oct 22;136(42):14726-9.
doi: 10.1021/ja508872q. Epub 2014 Oct 7.

Positive allostery in metal ion binding by a cooperatively folded β-peptide bundle

Jonathan P Miller  1 , Michael S Melicher, Alanna Schepartz
Affiliations

Positive allostery in metal ion binding by a cooperatively folded β-peptide bundle

Jonathan P Miller et al. J Am Chem Soc. .

Erratum in

Abstract

Metal ion binding is exploited by proteins in nature to catalyze reactions, bind molecules, and favor discrete structures, but it has not been demonstrated in β-peptides or their assemblies. Here we report the design, synthesis, and characterization of a β-peptide bundle that uniquely binds two Cd(II) ions in a distinct bicoordinate array. The two Cd(II) ions bind with positive allosteric cooperativity and increase the thermodynamic stability of the bundle by more than 50 °C. This system provides a unique, synthetic context to explore allosteric regulation and should pave the way to sophisticated molecular assemblies with catalytic and substrate-sensing functions that have historically not been available to de novo designed synthetic proteomimetics in water.

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Figures

Figure 1
Figure 1
(A) Ribbon diagram of the previously reported Zwit-YK β-peptide bundle structure determined by X-ray crystallography. The locations of the N- and C-termini of each strand are indicated by cyan and red coloring, respectively. Close-up of the potential two-coordinate Cd2+ binding site formed at the (B) parallel and (C) perpendicular helix interface in a model of the Zwit-YK-C octameric bundle. The perpendicular interface is rotated 40 degrees for clarity. (D) Primary sequence of Zwit-YK-C monomer.
Figure 2
Figure 2
Circular dichroism (CD) and sedimentation equilibrium analytical ultracentrifugation (SE-AU) analysis of β-peptide bundle formation by Zwit YK-C in TT buffer (5 mM Tris-Cl (pH 8), 1 mM TCEP). (A) Wavelength-dependent CD spectra of Zwit YK-C (25 °C) at concentrations between 1.6 and 200 μM. (B) Plot of the MRE at 210 nm as a function of [Zwit YK-C]T and fit to an ideal monomer-ocatamer equilibrium. (C) SE-AU analysis of Zwit-YK-C at 120 μM in TT buffer containing 100 mM NaCl, fit to a monomer-octamer equilibrium. (D) RMSD of the SE-AU fits as a function of n.
Figure 3
Figure 3
Plots illustrating Cd2+ binding by (A) β-YACAACA and (B–D) the Zwit YK-C β3-peptide bundle. (A) UV–vis difference spectra of β-YACAACA (200 μM) in the presence of high (200 μM) or moderate (50 μM) Cd2+, normalized to show the shift in LMCT signal. (B) UV–vis difference spectra of Zwit YK-C (100 μM) as the [Cd2+] varies between 0 and 75 μM. (C) Plot of absorbance at 245 nm of 50 μM Cd2+ as a function of added [Zwit YK-C]T showing a plateau at 4 equiv of [Zwit YK-C]T. (D) Temperature-dependent CD spectra illustrating cooperative unfolding of the Zwit YK-C bundle ([Zwit YK-C]T = 100 μM) both alone (Tm = 41.5 °C) and in the presence of 30 μM CdCl2 (Tm > 90 °C).
Figure 4
Figure 4
(A) Plot of absorbance (245 nm) of Zwit YK-C (100 μM) as a function of added [Cd2+]. A sigmoidal fit to the Hill equation is shown by the solid black curve, while a noncooperative fit is shown by the dashed curve. (B) Isothermal titration calorimetry (ITC) analysis of Cd2+ binding by the Zwit-TK-C β3-peptide bundle in 5 mM Tris, 1 mM TCEP (pH 8.1) at 25 °C. Data was fit to a one-site model in which n was an independent variable. The ITC output is shown in green; the integrated heat per injection in blue.
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