Peptide-equipped tobacco mosaic virus templates for selective and controllable biomineral deposition

Abstract

The coating of regular-shaped, readily available nanorod biotemplates with inorganic compounds has attracted increasing interest during recent years. The goal is an effective, bioinspired fabrication of fiber-reinforced composites and robust, miniaturized technical devices. Major challenges in the synthesis of applicable mineralized nanorods lie in selectivity and adjustability of the inorganic material deposited on the biological, rod-shaped backbones, with respect to thickness and surface profile of the resulting coating, as well as the avoidance of aggregation into extended superstructures. Nanotubular tobacco mosaic virus (TMV) templates have proved particularly suitable towards this goal: Their multivalent protein coating can be modified by high-surface-density conjugation of peptides, inducing and governing silica deposition from precursor solutions in vitro. In this study, TMV has been equipped with mineralization-directing peptides designed to yield silica coatings in a reliable and predictable manner via precipitation from tetraethoxysilane (TEOS) precursors. Three peptide groups were compared regarding their influence on silica polymerization: (i) two peptide variants with alternating basic and acidic residues, i.e. lysine-aspartic acid (KD) x motifs expected to act as charge-relay systems promoting TEOS hydrolysis and silica polymerization; (ii) a tetrahistidine-exposing polypeptide (CA4H4) known to induce silicification due to the positive charge of its clustered imidazole side chains; and (iii) two peptides with high ZnO binding affinity. Differential effects on the mineralization of the TMV surface were demonstrated, where a (KD) x charge-relay peptide (designed in this study) led to the most reproducible and selective silica deposition. A homogenous coating of the biotemplate and tight control of shell thickness were achieved.

Keywords: biomineralization; charge-relay system; peptide; silica; tobacco mosaic virus (TMV).

Figure 1

Schematic representation of the chemical modification and mineralization of tobacco mosaic virus (TMV) nucleoprotein nanotubes. (a) Genetically engineered virus particles with thousands of surface-exposed amino groups of lysine residues (TMV_Lys) served as biotemplates for chemical conjugation reactions. (b) Hetero-bifunctional linker molecules (succinimidyl-(N-maleimidopropionamido) ester, SM(PEG)₄) were coupled to TMV_Lys via N-hydroxysuccinimide (NHS) ester-mediated crosslinking with lysine primary amines, yielding amide bonds. (c) Mineralization-affecting peptides were conjugated to the maleimide-activated SM(PEG)₄ linker portion via the sulfhydryl groups of their terminal cysteine residues, yielding stable thioether linkages. The resulting functionalized TMV templates fashioned with a dense peptide coating were (d) subjected to silica mineralization via hydrolysis and condensation of a tetraethoxysilane (TEOS) precursor in solution (mechanism indicated).

Figure 2

Gel electrophoretic analysis of chemically modified TMV–Lys particles. (a) SDS-PAGE shows retarded bands of CPs modified with the linker SM(PEG)₄ (diamond, PEG), or after coupling SM(PEG)₄ and different peptides (stars, peptides as indicated above), compared to unmodified CP_Lys (triangle, Lys). (b) Peptide-equipped TMV–Lys particles exhibiting different separation patterns during native agarose gel electrophoresis, indicating various states of head-to-tail aggregation in combination with distinct negative overall charges. Moieties exposed on the TMV templates are indicated (abbreviations as in Table 2). Numbers on the right: approximate numbers of TMV particles in head-to-tail aggregates (in relation to lane “TMV–PEG”).

Figure 3

Zeta potential of bare and chemically modified TMV–Lys particles in ddH₂O or 30 mM Tris-HCl pH 8.0, respectively (modifications of TMV rods indicated above).

Figure 4

SiO₂ deposition reactions using functionalized and non-modified TMV templates, as indicated. (a) Images of sedimented products, and (b) corresponding SEM analysis. TMV–Lys-template (or water control) solutions were mixed with absolute EtOH (99.9%) and TEOS in a 4:4:1 volume ratio. Reaction products were sedimented by centrifugation (after 7 days of incubation in (a) or 10 days in (b)), resuspended in ddH₂O and prepared for SEM (for details, refer to text).

Figure 5

Time-resolved monitoring of silica shell growth on TMV–KD10 templates: TEM analysis of non-stained specimens, after the reaction times indicated above. Total average diameters (Ø ± standard deviations) of mineralized TMV–KD10-hybrids were determined from 11–15 randomly selected nanorod products collected between one and twelve days of incubation.

Figure 6

ToF-SIMS analysis for determination of silica deposition. TMV–KD10 with TEOS (blue) and without TEOS (green), TMV_wt with TEOS (red) and without TEOS (purple) after ten days of incubation. The peak at m/z 27.97 indicates Si, the peak at m/z 28.02 CH₂N⁺, and the peak at m/z 28.03 C₂H₄⁺. For TMV–KD10 with TEOS and TMV_wt with TEOS, the decrease of the CH₂N⁺ peak, indicating peptide/protein components, is an indirect effect of the mineralization, shielding the soft-matter surface of biotemplate particles.