Genetically programmable thermoresponsive plasmonic gold/silk-elastin protein core/shell nanoparticles

Abstract

The design and development of future molecular photonic/electronic systems pose the challenge of integrating functional molecular building blocks in a controlled, tunable, and reproducible manner. The modular nature and fidelity of the biosynthesis method provides a unique chemistry approach to one-pot synthesis of environmental factor-responsive chimeric proteins capable of energy conversion between the desired forms. In this work, facile tuning of dynamic thermal response in plasmonic nanoparticles was facilitated by genetic engineering of the structure, size, and self-assembly of the shell silk-elastin-like protein polymers (SELPs). Recombinant DNA techniques were implemented to synthesize a new family of SELPs, S4E8Gs, with amino acid repeats of [(GVGVP)4(GGGVP)(GVGVP)3(GAGAGS)4] and tunable molecular weight. The temperature-reversible conformational switching between the hydrophilic random coils and the hydrophobic β-turns in the elastin blocks were programmed to between 50 and 60 °C by site-specific glycine mutation, as confirmed by variable-temperature proton NMR and circular dichroism (CD) spectroscopy, to trigger the nanoparticle aggregation. The dynamic self-aggregation/disaggregation of the Au-SELPs nanoparticles was regulated in size and pattern by the β-sheet-forming, thermally stable silk blocks, as revealed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The thermally reversible, shell dimension dependent, interparticle plasmon coupling was investigated by both variable-temperature UV-vis spectroscopy and finite-difference time-domain (FDTD)-based simulations. Good agreement between the calculated and measured spectra sheds light on design and synthesis of responsive plasmonic nanostructures by independently tuning the refractive index and size of the SELPs through genetic engineering.

Figure 1

Schematic diagram of the NTA-Ni²⁺ functionalized Au NPs for surface recognition of the thermoresponsive S4E8G protein polymers, facilitated by an N-terminal polyhistidine tag. The responsive self-assembly characteristics of Au-S4E8Gs are based on the thermally reversible conformational switching in the elastin-like block between the hydrophilic random coil conformation and the hydrophobic β-spiral adopting repetitive type II β-turns.^−,

Figure 2

(a) Molecular design and construction of the recombinant S4E8G protein polymers consisting of tandemly repeated elastin-like pentapeptide sequence GVGVP and silk-like hexapeptide sequence GAGAGS. The trigger temperatures for thermal response of the Au-S4E8G NPs were genetically engineered to between 50 and 60 °C (b). Identity (b) and purity of the recombinant S4E8Gs, containing four different numbers of silk-elastin-like repeats: 3-, 8-, 13-, and 18-mer, were characterized by mass spectrometry and (c) SDS-PAGE.

Figure 3

Molecular origin of the reversible thermal response of the recombinant S4E8G protein polymers. A heating-induced conformational transition in the elastin-like domains of S4E8G-18-mer, from random coil to type II β-turn, was revealed by variable-temperature CD spectroscopy. (a) S4E8G-18-mers (0.3 mg/mL in H₂O) at low temperatures were found to adopt random coil-dominated structures, containing small amounts of β-conformations. The inset shows reference CD spectra of regenerated B. mori silk (0.05 wt % in H₂O), adopting 100% random coil at pH 10 and containing about 5% of β-sheets at pH 6. The evolution of a positive ellipticity band with a maximum at 210 nm during heating confirms the growth of type II β-turn. Note that an isodichroic point occurred at ∼213 nm, implying two-state solution behavior, and that the β-structures in the silk-like domains were not affected by temperature. (b) Thermally induced conversion between random coil and β-turn in the elastin-like domains was demonstrated to be rapidly reversible in the hysteresis loops.

Figure 4

Heating-induced conformational switching in S4E8G-18-mer was further studied by variable-temperature ¹H NMR spectroscopy. (a, b) Heating-induced changes (broadening, intensity decrease, and downfield shifts) in the amide proton NMR signals verified the decrease of chain mobility in S4E8G-18-mers (1 mg/mL in D₂O, containing the external standard HMDS) at higher temperatures.^, (c) Thermal activation of the proton exchange, for example, between the elastin glycine NH and H₂O, determined from the slope of the temperature dependence of NH chemical shifts, was found weaker above the transition temperature, suggesting that the formation of hydrogen bonds in the elastin-like domains.

Figure 5

Thermally reversible assembly of plasmonic NPs programmed by genetically engineered S4E8G proteins. (a) TEM images of Au-S4E8G-18mer NPs (1.2 nM in 10 mM PBS at pH 7.4) in individual form at 25 °C and (b) in aggregated form when heated to 60 °C. The square-like shape of (b) the Au-S4E8G NP aggregate was an indication for the presence of β-strands/β-sheets in the silk blocks of S4E8G, as also confirmed by CD. The thermal stability of the silk blocks, as a resistance mechanism opposing the temperature-induced coacervation, functionalized to regulate the aggregate size.

Figure 6

DLS studies on the reversible, temperature dependent aggregation behavior of the four Au-S4E8G NPs (all at 0.1 nM) in PBS buffer (10 mM, pH 7.4).

Figure 7

On/off thermal switching between the individual and collective plasmon resonances in the Au-S4E8G NPs. (a) Variable-temperature UV–vis extinction spectra of the Au-S4E8G-18-mer NPs (1.0 nM in 10 mM PBS at pH 7.4) revealed a peak extinction at ∼522 nm for the ruby red solution as-prepared at room temperature. When heated to 60 °C, the absorption peak red-shifted to ∼545 nm, and the color turned to pink-red (see the inset). (b) Simulated extinction spectra were in good agreement with the experimental results. (c) Thermal cycles of the Au-S4E8G-3-mer and -18-mer NPs between 25 and 60 °C were monitored by UV–vis spectroscopy showing good reversibility.