Understanding Self-Assembly of Silica-Precipitating Peptides to Control Silica Particle Morphology

Abstract

The most advanced materials are those found in nature. These evolutionary optimized substances provide highest efficiencies, e.g., in harvesting solar energy or providing extreme stability, and are intrinsically biocompatible. However, the mimicry of biological materials is limited to a few successful applications since there is still a lack of the tools to recreate natural materials. Herein, such means are provided based on a peptide library derived from the silaffin protein R5 that enables rational biomimetic materials design. It is now evident that biomaterials do not form via mechanisms observed in vitro. Instead, the material's function and morphology are predetermined by precursors that self-assemble in solution, often from a combination of protein and salts. These assemblies act as templates for biomaterials. The RRIL peptides used here are a small part of the silica-precipitation machinery in diatoms. By connecting RRIL motifs via varying central bi- or trifunctional residues, a library of stereoisomers is generated, which allows characterization of different template structures in the presence of phosphate ions by combining residue-resolved real-time NMR spectroscopy and molecular dynamics (MD) simulations. Understanding these templates in atomistic detail, the morphology of silica particles is controlled via manipulation of the template precursors.

Keywords: biomineralization; molecular dynamics; peptide self-assembly; silica particles; silica precipitation.

Scheme 1

A) Overview of the R5 peptide and RRIL peptide dimers. B) Library of d‐ and l‐amino acids containing isopeptide RRIL/rrill dimers explored here. Lowercase letters indicate d‐amino acids. Central amino acids used here are l‐ or d‐Dap = 2,3‐diaminopropionic acid in peptides 1, 5, 8, and 11, βAla = β‐alanine in peptides 2 and 12, l‐ or d‐Orn = ornithine in peptides 3, 6, 9, and 13 and l‐ or d‐Lys = lysine in peptides 4, 7, 10, and 14.

Figure 1

Comparison of the peptides 1–4 with respect to silica particle precipitation. The sidechain‐attached rrill (green) as well as the C‐terminal RRIL (blue) sequences and the stereochemistry of the linker are kept constant, whereas the linker length was varied by using l‐Dap, βAla, l‐Orn, or l‐Lys as central residue.

Figure 2

Comparison of the peptides 2, 5–7 with respect to silica particle precipitation. The sidechain‐attached rrill (green) as well as the C‐terminal RRIL (blue) sequences and the stereochemistry of the linker are kept constant, whereas the linker length was varied by using d‐Dap, βAla, d‐Orn, or d‐Lys as central residues.

Figure 3

Comparison of peptides 11–14 with respect to silica particle precipitation. The sidechain‐attached RRILL (green) as well as the C‐terminal rril (blue) sequences and the stereochemistry have been switched to generate the enantiomers of peptides 5, 6, and 7.

Figure 4

Comparison of peptides 8–10 and 12 with respect to silica particle precipitation. The sidechain‐attached RRILL (green) as well as the C‐terminal rril (blue) sequences and the stereochemistry have been switched to generate the enantiomers of peptides 1, 3, and 4.

Figure 5

a) H^N‐region of ¹H‐¹H TOCSY spectra of rrillk*RRIL 7 in water (blue) and PBS (red). The resonance assignment is indicated. The resonance of K⁶ is heavily broadened due to rapid proton exchange. Dissolution in PBS leads to a disappearance of most resonances. b) H^N/H^α‐region of ¹H‐¹H TOCSY (red) and NOESY (purple) spectra of 7 in PBS. Residues i³, l⁴, I⁹, and the side chain of R⁷ indicate solvent exposure through NOE and/or exchange‐based cross‐peaks with the water resonance. c) H^N/H^α‐region of ¹H—¹H TOCSY (red) and NOESY (purple) spectra of rrillK*RRIL 4 in PBS. In contrast to 7, no water interaction can be observed. d) Simulated structure of 7 in PBS. The l‐ and d‐blocks are indicated by the red/blue color code. N‐ and C‐termini are labeled. Note the hinge involving the k⁶ isopeptide bond. e) Simulated structure of 4 in PBS. f) Aggregate structure found in MD simulations of 5 copies of 7 in PBS. The gray circles indicate salt bridges formed by the phosphate ions and positively charged arginines. g) Same as in panel (f), but for 4. Besides the salt bridges, hydrophobic side‐chain contacts (yellow circles) cause compaction of the aggregates.

Figure 6

Real‐time monitoring of 4 precipitation. a) Residue‐resolved intensity time‐traces after addition of the silicic acid. Initially, the signals lose intensity due to the precipitation event. After ≈15 min all resonances reappear, pointing towards a weak resolubilization effect. b) Comparison of the initial intensity decay for residues L⁵ and I⁹. The C‐terminal isoleucine loses its signal slower than the central leucine. c) Cooperativity parameter for all visible residues as determined by fitting a sigmoidal function to the first 5 min of the time traces in (a). The higher the value, the slower the onset of the precipitation event. Residues I⁹ and L¹⁰ show higher values, i.e., a slower signal loss.