Sequence Changes Modulate Peptoid Self-Association in Water

Abstract

Peptoids, N-substituted glycine oligomers, are a class of diverse and sequence-specific peptidomimetics with wide-ranging applications. Advancing the functional repertoire of peptoids to emulate native peptide and protein functions requires engineering peptoids that adopt regular secondary and tertiary structures. An understanding of how changes to peptoid sequence change structural features, particularly in water-soluble systems, is underdeveloped. To address this knowledge gap, five 15-residue water-soluble peptoids that include naphthalene-functionalized side chains were designed, prepared, and subjected to a structural study using a palette of techniques. Peptoid sequence designs were based on a putative amphiphilic helix peptoid bearing structure-promoting (S)-N-(1-naphthylethyl)glycine residues whose self-association in water has been studied previously. New peptoid variants reported here include sequence changes that influenced peptoid conformational flexibility, functional group patterning (amphiphilicity), and hydrophobicity. Peptoid structures were evaluated and compared using circular dichroism spectroscopy, fluorescence spectroscopy, and size exclusion chromatography. Spectral data confirmed that sequence changes alter peptoids' degree of assembly and the organization of self-assembled structures in aqueous solutions. Insights gained in these studies will inform the design of new water-soluble peptoids with regular structural features, including desirable higher-order (tertiary and quaternary) structural features.

Keywords: circular dichroism (CD) spectroscopy; fluorescence spectroscopy; peptidomimetic; peptoid; self-association; size exclusion chromatography.

Figure 1

Structure of peptoid 1, a 15-residue, water-soluble peptoid with putative amphiphilic helix structure that self-associates in aqueous solution.

Figure 2

Peptoid synthesis and structures used in this study. (A) Peptoid synthesis following the submonomer method. DIC, diisopropylcarbodiimide. (B) Structures of all peptoids prepared. (C) Helix wheel representations of peptoids studied. Ar, aromatic residue (Ns1npe, Ns2npe, or Nspe); Nsce, negative charge; Nae, positive charge.

Figure 3

Changes to fluorescence emission intensity of 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) as influenced by increasing concentrations of 1–5. Spectra were collected for solutions of 50 μM 1,8-ANS plus varied concentrations of peptoid in Tris-buffered saline (TBS buffer) at room temperature.

Figure 4

Circular dichroism (CD) spectra of peptoids 1 (black squares), 2 (red circles), 3 (blue triangles, point up), 4 (gray triangles, point down), 5 (light blue diamonds). Peptoids were 40 μM in 5 mM Tris buffer, pH 7.5, and spectra were acquired at 20°C. Inset shows the near-UV region of the spectrum (260–320 nm).

Figure 5

Far-UV circular dichroism (CD) spectra of 1–5 at varied temperatures ranging from 2 to 98°C. All peptoids were 50 μM in 5 mM Tris buffer, pH 7.5.

Figure 6

Fluorescence emission spectra of 1–5. Spectra were recorded at room temperature for peptoid solutions in 5 mM Tris buffer, pH 7.5.

Figure 7

Comparisons of far-UV circular dichroism (CD) spectra of 1–5 in three different aqueous buffers. Peptoids were 50 μM in the indicated buffer, and spectra were acquired at 20°C.

Figure 8

Comparisons of fluorescence emission spectra of 1–5 in three different aqueous buffers. Peptoids were 40 μM in the indicated buffer, and spectra were acquired at room temperature.