Cooperative self-assembly of peptide gelators and proteins

Abstract

Molecular self-assembly provides a versatile route for the production of nanoscale materials for medical and technological applications. Herein, we demonstrate that the cooperative self-assembly of amphiphilic small molecules and proteins can have drastic effects on supramolecular nanostructuring of resulting materials. We report that mesoscale, fractal-like clusters of proteins form at concentrations that are orders of magnitude lower compared to those usually associated with molecular crowding at room temperature. These protein clusters have pronounced effects on the molecular self-assembly of aromatic peptide amphiphiles (fluorenylmethoxycarbonyl- dipeptides), resulting in a reversal of chiral organization and enhanced order through templating and binding. Moreover, the morphological and mechanical properties of the resultant nanostructured gels can be controlled by the cooperative self-assembly of peptides and protein fractal clusters, having implications for biomedical applications where proteins and peptides are both present. In addition, fundamental insights into cooperative interplay of molecular interactions and confinement by clusters of chiral macromolecules is relevant to gaining understanding of the molecular mechanisms of relevance to the origin of life and development of synthetic mimics of living systems.

Scheme 1. (a) Low Molecular Weight Hydrogelators (Fmoc-Dipeptides), (b) Protein Structures of β-LG/BSA Used in the Study and Their Hydropathy Indices (hI) and Isoelectric Points (pI), (c) Supramolecular Assembly of Peptides Resulting in Fibrous Structures and Proteins Yielding Mesoscale Fractal Clusters

The hydropathy index (hI) was calculated using “grand average hydropathy (GRAVY)”.

Figure 1

Static light scattering intensity patterns for different concentrations of β-LG and BSA in 100 mM phosphate buffer at pH 8 and room temperature.

Figure 2

Spectroscopic characterization of Fmoc-dipeptides self-assembly with/without the addition of various concentrations of β-LG: (a) CD spectra of Fmoc-YL self-assembly. (b) Effect of protein clusters on ellipticity (at wavelength of 302 nm due to Fmoc-group) for different Fmoc- dipeptides. (c) Fluorescence spectra of Fmoc-YL self-assembly. (d) Effect of protein clusters on fluorescence intensity for different Fmoc-dipeptides.

Figure 3

Spectroscopic characterization of Fmoc-dipeptides self-assembly with/without the addition of various concentrations of BSA: (a) CD spectra of Fmoc-YL self-assembly. (b) Effect of protein clusters on ellipticity (at wavelength of 302 nm due to Fmoc-group) for different Fmoc- dipeptides. (c) Fluorescence spectra of Fmoc-YL self-assembly. (d) Effect of protein clusters on fluorescence intensity for different Fmoc-dipeptides.

Figure 4

Fluorescence spectra of Fmoc-YL (0.06 mM) in the presence of various concentrations of BSA and β-LG in 100 mM phosphate buffer at pH 8 at room temperature to calculate the binding constants.

Figure 5

(a) Rheological characterization of fibrous hydrogels formed in the presence of various concentrations of β-LG: Typical frequency sweep patterns of Fmoc-YL hydrogels showing storage and loss modulus, G′ and G″. (b) Storage modulus G′ for hydrogels measured 24 h after of gelation for various Fmoc-dipeptides. Atomic force microscopy images of hydrogels (for YL, YN, and YS) without protein (c–e) and with 0.2 wt % β-LG (f–h): scale bar, 2 μm. The storage moduli G′ for all hydrogels with different amino acid sequences and different concentrations of proteins were determined by frequency sweeps and are summarized in Table 2

Figure 6

(a) Rheometric characterization: Typical frequency sweep patterns of Fmoc-YL hydrogels with added BSA showing storage modulus, G′ and loss modulus, G″. (b) The modulation of storage modulus G′ of the hydrogels with added different concentrations of BSA. Atomic force microscopy images of hydrogels with added 0.2 wt % BSA for YL (c), YN (d), and YS (e): scale bar, 2 μm.