Residue-Specific Solvation-Directed Thermodynamic and Kinetic Control over Peptide Self-Assembly with 1D/2D Structure Selection

Abstract

Understanding the self-organization and structural transformations of molecular ensembles is important to explore the complexity of biological systems. Here, we illustrate the crucial role of cosolvents and solvation effects in thermodynamic and kinetic control over peptide association into ultrathin Janus nanosheets, elongated nanobelts, and amyloid-like fibrils. We gained further insight into the solvation-directed self-assembly (SDSA) by investigating residue-specific peptide solvation using molecular dynamics modeling. We proposed the preferential solvation of the aromatic and alkyl domains on the peptide backbone and protofibril surface, which results in volume exclusion effects and restricts the peptide association between hydrophobic walls. We explored the SDSA phenomenon in a library of cosolvents (protic and aprotic), where less polar cosolvents were found to exert a stronger influence on the energetic balance at play during peptide propagation. By tailoring cosolvent polarity, we were able to achieve precise control of the peptide nanostructures with 1D/2D shape selection. We also illustrated the complexity of the SDSA system with pathway-dependent peptide aggregation, where two self-assembly states ( i.e., thermodynamic equilibrium state and kinetically trapped state) from different sample preparation methods were obtained.

Keywords: 2D structures; fibrils; pathway dependence; peptide solvation; self-assembly.

Figure 1

(a) Asymmetric peptide (F6C11) containing one glutamic acid (Glu) at the C-terminus, two glutamic acids (Glu₂) at the N-terminus, six phenylalanine residues (Phe₆), and a hydrocarbon chain (C11). (b) Schematic of solvation-directed self-assembly with 1D and 2D shape selectivity: formation of 2D nanosheets in aqueous solution is proposed to be guided by hydrogen bonding in the x-axial direction, π–π stacking in the y-axial direction, and hydrophobic effect in both directions. Amyloid-like fibrils are the thermodynamically favored products in the presence of cosolvents (i.e., n-propanol), where the strong solvation effect of aromatic and alkyl groups by n-propanol are supposed to weaken the π–π stacking and hydrophobic effect.

Figure 2

(a–d) Transmission electron microscopy (TEM) images and (e–h) histogram profile of length/diameter ratio (L/D) of F6C11 (0.25 mM) supramolecular structures with increasing methanol concentrations: (a,e) 0%; (b,f) 5%; (c,g) 10%; (d,h) 15% methanol. The nanosheet-to-nanofibril (2D-to-1D) structural evolution occurs with an increase in methanol concentration. (i) L/D ratio of peptide nanostructures determined from TEM images (mean ± SD). (j) Optical density (OD) and (k) ThT fluorescence intensity of peptide solution. (l) Circular dichroism (CD) spectra showing the disordering of β-sheet secondary structures with increased methanol. Scale bar: (a–c) 1 μm and (d) 500 nm.

Figure 3

TEM images showing the morphological changes of F6C11 self-assembly (0.25 mM) in the presence of different alcohols (5%): (a) ethanol, (b) n-propanol, (c) n-butanol, (d) ethylene glycol. Peptide nanofibrils were the thermodynamically favored morphology in the presence of less polar cosolvents like n-butanol and n-propanol, whereas nanosheets were the dominant morphology when the cosolvent is more polar (e.g., ethylene glycol). A cosolvent with intermediate polarity (e.g., ethanol) drives F6C11 self-assembly into elongated nanobelts. Scale bar: (a,d) 1 μm; (b,c) 200 nm.

Figure 4

Distributions of the fraction solvent-accessible surface area (SASA) of the six phenylalanine (Phe₆) residues in individual F6C11 molecules covered by (a) methanol, (b) ethanol, (c) n-propanol, and (d) n-butanol. The average SASA of the Phe₆ region is ∼1000 Ų, and on average, 131, 151, 208, 379 Ų of the Phe₆ surface is covered by methanol, ethanol, n-propanol, and n-butanol, respectively. (e) Average percentage of SASA covered by organic solvents for F6C11 molecules immersed in water–alcohol mixtures. Glu₂: two glutamic acid at the N-terminus; Phe₆: phenylalanine; C11: alkyl group; Glu: glutamic acid at the C-terminus. Note: numbers in (e) are slightly different by ±0.2% from those in (a–d) due to rounding in figures (a–d).

Figure 5

(a) F6C11₁₀ protofibril shown as a surface with atoms colored according to charge with a color range of −0.5 (red), 0.0 (white) and +0.5 (blue). Exemplar snapshot of the F6C11₁₀ protofibril immersed in (b) water–methanol and (c) water–propanol mixture. Organic solvent molecules within 4 Å of the F6C11₁₀ are shown in cyan. (d–g) Distributions of the fraction solvent-accessible surface area (SASA) of the Phe₆ residues in the protofibril covered by (d) methanol, (e) ethanol, (f) n-propanol, and (g) n-butanol with fitted normal distributions. (h) Average percentage of SASA covered by organic solvents. Glu₂: two glutamic acid at the N-terminus; Phe₆: phenylalanine; C11: alkyl group; Glu: glutamic acid at the C-terminus.

Figure 6

TEM images showing the structural evolution of F6C11 (0.25 mM) self-assembly in the presence of aprotic solvents (5 v/v%): (a) formamide, (b) N-methyl formamide, (c) N,N-dimethylformamide, (d) N,N-dimethylacetamide. Noteworthy, the decrease of polarity from formamide (E_T^N = 0.775), N-methyl formamide (E_T^N = 0.722), N,N-dimethylformamide (E_T^N = 0.386), and N,N-dimethylacetamide (E_T^N = 0.377) leads to a structural transition from nanosheets to elongated nanobelts and nanofibrils. Scale bar: (a–c) 500 nm, (d) 250 nm.

Figure 7

(a) Schematic Gibbs free energy landscape of solvation-directed self-assembly, where Pathway I led to a thermodynamic equilibrium state corresponding to the formation of fibrils (state 1), and Pathway II resulted in a kinetically trapped state corresponding to nanosheets (state 2). Pathway I: aggregation of F6C11 monomers was completed in aqueous solution containing cosolvents. Pathway II: cosolvents were added to preformed F6C11 nanosheets. (c–h) TEM images showing the amyloid-like fibrils in state 1, following Pathway I (c–f), and nanosheets in state 2, following Pathway II (g–j). Cosolvents were used to induce structural evolution of F6C11 under two pathways: (c,g) n-propanol; (d,h) isopropyl alcohol; (e,i) DMF; (f,j) THF. Scale bar: (a) 200 nm; (d–f) 500 nm; (g–j) 1 μm. (k–n) ThT fluorescence in the F6C11 peptide solution showing the strengths of molecular packing varied in different SDSA pathways: (k) n-propanol; (l) isopropyl alcohol; (m) DMF; (n) THF.