A Matter of Charge: Electrostatically Tuned Coassembly of Amphiphilic Peptides

Abstract

Coassembly of peptide biomaterials offers a compelling avenue to broaden the spectrum of hierarchically ordered supramolecular nanoscale structures that may be relevant for biomedical and biotechnological applications. In this work coassemblies of amphiphilic and oppositely charged, anionic and cationic, β-sheet peptides are studied, which may give rise to a diverse range of coassembled forms. Mixtures of the peptides show significantly lower critical coassembly concentration (CCC) values compared to those of the individual pure peptides. Intriguingly, the highest formation of coassembled fibrils is found to require excess of the cationic peptide whereas equimolar mixtures of the peptides exhibited the maximum folding into β-sheet structures. Mixtures of the peptides coassembled sequentially from solutions at concentrations surpassing each peptide's intrinsic critical assembly concentration (CAC), are also found to require a higher portion of the cationic peptide to stabilize hydrogels. This study illuminates a systematic investigation of oppositely charged β-sheet peptides over a range of concentrations, in solutions and in hydrogels. The results may be relevant to the fundamental understanding of such intricate charge-driven assembly systems and to the formulation of peptide-based nanostructures with diverse functionalities.

Keywords: charged peptides; coassembly; peptide biomaterials; peptide hydrogels; β‐sheet peptides.

Figure 1

PM coassemblies in HEPES pH = 7.4 solutions. List of the model peptides and their sequences. A) Critical Assembly Concentration (CAC, blue) of the peptides in separate (0%, 100%) and Critical Coassembly Concentration (CCC, red) of the PM as function of the An‐FD %, measured by ANS fluorescence (see also Figure S1, Supporting Information). Mixtures were prepared by mixing the solutions of each peptide in its unassembled state, at concentrations ranging from 1 µm to 1 mm, at different volumetric ratios followed by the addition of ANS and then subjected to fluorescence measurements. B) ANS fluorescence of 0.3 mm PM, as a function of An‐FD or Sc‐FD %. Data (dots) were fitted to a Lorentzian function (lines) indicating the maximal signal is at 40% ratio of both these anionic peptides. Inset shows representative pictures of the PM solutions showing mild turbidity. C) CD spectra showing normalized ellipticity versus wavelength, of 0.3 mm Cat‐FK, An‐FD, and their PM at selected An‐FD %. D) Ratio of β‐sheet ellipticity minimum (218 nm) to that of the predominantly PPII conformations (203 nm) as a function of An‐FD % for different PM and the pure peptides. E) CD spectra of PM composed of Sc‐FD and Cat‐FK at a total peptide concentration of 0.1 mm since higher concentrations could not be measured due to excessive aggregation and turbidity that is affecting the spectra at the shorter wavelengths. The broken black line represents PM of 50% An‐FD as positive control. F) CD spectra of 0.3 mm PM of Sc‐FK and An‐FD, showing increased β‐sheet content up to the 50% An‐FD, see inset representing ratio of ellipticities as in D. For panels A, B, and D, the values represent an average ± SD, n = 3.

Figure 2

Fluorescently labeled PM coassemblies formed at a total concentration of 0.1 mm. Each row shows a certain PM, represented by the An‐FD % in the mixture. FITC‐labeled Cat‐FK solution and Rhodamine‐red labelled An‐FD solution were mixed and incubated at a total concentration of 0.1 mm in HEPES 50 mm, pH = 7.4 (each peptide contained 1% mol/mol of its covalently‐labelled fluorescent version). The samples were imaged separately for the FITC fluorescence (left), Rhodamine‐red fluorescence (middle), and the merged image of the same area were created using ImageJ software to demonstrate the coassembly of the two peptides (right). The bars correspond to 10 µm.

Figure 3

Cryo‐TEM images of An‐FD, Cat‐FK, and PM. A) Below the CAC, each peptide at 0.05 mm does not show notable assembly. In contrast, PM of 50% An‐FD at the same concentration, above the CCC, shows nest‐morphology of short, entangled fibrils. Inset shows a higher magnification of the 50% PM fibrils. B) Above the CAC, each peptide at 22.7 mm (4% w/v) shows fibril formation. Insets show higher magnification of the fibrils. Cryo‐TEM image for PM of 50% at this total concentration could not be achieved due to excessive aggregation (Cryo‐SEM analysis is shown in Figure 7). Figure on the right shows the frequency of fibrils widths above the CAC of the Cat‐FK (0%) and the An‐FD (100%), in which the average widths are 7.6 ± 1.1 and 4.4 ± 0.5 nm (Avg. ± SD, n = 104 and 98, respectively) for An‐FD and Cat‐FK, respectively. Scale bars correspond to 200 nm (white) and in inset to 50 nm (black).

Figure 4

ThT, CR, and charge of 0.3 mm PM coassemblies. A) ThT signal (dots) fitted to a sigmoidal function (line). B) ζ‐potentials (dots connected by a line to guide the eye). Sample are presented as average±SD, n = 3. C) CR absorbance (dots) and lines to guide the eye. The spectra are presented in Figure S3 (Supporting Information). D) Scheme representing the fibrils formed by An‐FD (red color arrow) and Cat‐FK (blue color arrow) coassemblies, arranged along an arrow that highlights the fibril's charge. The distribution of both peptides within the PM was assessed by MALDI‐ToF that may support the presence of heteromeric oligomers (see Figure S6, Supporting Information).

Figure 5

The effect of pH on PM structures. A) Calculated formal charge of the PM as function of the pH considering the amino acids in the sequence (the bold line of 50% An‐FD refers to the samples of part B). B) CD spectroscopy of 50% An‐FD (molar ratio of 1:1 An‐FD: Cat‐FK) at different pH values showing a combination of β‐sheet and PPII structures (see inset). In addition, secondary structure analysis of the PM which was performed by Fourier Transform Infrared Spectroscopy (FTIR) showed amide‐I vibration typical to β‐sheet structure (Figure S8, Supporting Information).

Figure 6

ITC measurements of Cat‐FK titrated into An‐FD. A) Titration of Cat‐FK into An‐FD (pink), Sc‐FD (turquoise) solutions and to a buffer (gray). B) Enthalpy versus mole‐ratio (dots) with a fit to unspecific single‐type interactions' model (color scheme as in A). Table inset shows the parameters fitted to Cat‐FK titrated into An‐FD (pink curve). Data was fitted using NanoAnalyze software, to the independent model ± confidence interval. *The entropy and free energy values are derived from the fitted model rather than from the collected points.

Figure 7

PM above the CAC. A) Vials with PM at different % An‐FD with such turned upside down to demonstrate hydrogel formation. B) Cryo‐SEM images of hydrogel (33%) and PM solutions (50 and 5% An‐FD); Bar corresponding to 200 nm.

Figure 8

Structural analysis of PM by XRD and SAXS. A) XRD pattern of lyophilized An‐FD, Cat‐FK, and their PM, at a total concentration above the CAC, 22.7 mm, B) SAXS of these pure peptides and PM at 22.7 mm (above CAC). C) A scheme of the proposed unit cell comprising the fibrils; inset Table assigning the detected repeat distances to Miller indices. D) A scheme of the PM fibrils above the CAC (including cryo‐TEM images from Figure 3, of the pure peptides at 22.7 mm, and cryo‐SEM image of 33% An‐FD gel; bar corresponds to 50 nm). The proposed structure is based on the EM‐structures and the lamellar‐features seen in the SAXS.

Figure 9

The main pathways in coassembly of the charged β‐sheet forming peptides Cat‐FK and An‐FD. Above the CCC and below the CAC (right side of the scheme) the unordered peptides can undergo cooperative heteromeric coassembly, particularly including hydrophobic interactions and formation of β‐sheet (seen in Figure 1; Figure S3–S5, Supporting Information). Maximal assembly to β‐sheet is achieved in contribution of both peptides (Figures S3, S5, and S7, Supporting Information), while excess of either of them can influence the surface charge of the fibril and dominate the outer layer of the assembly (Figure 4; Figure S6, Supporting Information). Incorporation of scrambled sequence leads to entropically‐driven disruptive coassembly, avoiding the fold into precise ordered structure (down arrow, based on Figures 1 and 6; Figure S6, Supporting Information). Above the CAC (left side of the scheme) the peptides first undergo homomeric self‐assembly (based on Figures 1, 3, and 8) which is followed by their orthogonal coassembly into either viscous solution or a hydrogel phase (as seen in Figures 7 and 8; Figure S10, Supporting Information).