Genetically Engineered Liposwitch-Based Nanomaterials

Abstract

Fusion of intrinsically disordered and globular proteins is a powerful strategy to create functional nanomaterials. However, the immutable nature of genetic encoding restricts the dynamic adaptability of nanostructures postexpression. To address this, we envisioned using a myristoyl switch, a protein that combines allostery and post-translational modifications─two strategies that modify protein properties without altering their sequence─to regulate intrinsically disordered protein (IDP)-driven nanoassembly. A typical myristoyl switch, allosterically activated by a stimulus, reveals a sequestered lipid for membrane association. We hypothesize that this conditional exposure of lipids can regulate the assembly of fusion proteins, a concept we term "liposwitching". We tested this by fusing recoverin, a calcium-dependent myristoyl switch, with elastin-like polypeptide, a thermoresponsive model IDP. Biophysical analyses confirmed recoverin's myristoyl-switch functionality, while dynamic light scattering and cryo-transmission electron microscopy showed distinct calcium- and lipidation-dependent phase separation and assembly. This study highlights liposwitching as a viable strategy for controlling DP-driven nanoassembly, enabling applications in synthetic biology and cellular engineering.

Figure 1

Schematic of our strategy for integrating post-translational modifications and allostery as mechanisms that drive external stimulus-responsive IDP-driven nanoassembly. Diagram of the constitutive domains: recoverin (R) as a myristoyl switch, ELP (E) as a thermoresponsive coil protein, and RE fusion. Recoverin transitions from a lipid-sequestered state to a lipid-exposed state upon Ca²⁺ binding (PDB ids 1IKU and 1JSA), and ELP undergoes temperature-induced aggregation.

Figure 2

Structural and functional integrity of recoverin in RE fusions. (a,b) Far-UV CD spectra reveal an α-helical structure in recoverin (Rc) and a mixture of α-helical (from R-domain) and random coil (from E-domain) structures in RE in apo form, unaffected by myristoylation (±m). Error bars represent standard deviations from two replicates. (c) Tryptophan fluorescence is red-shifted in both Rc and RE, observed only with myristoylation (+m) and calcium (+Ca²⁺), consistent with the displacement of the lipid from recoverin’s core. The dashed line at 345 nm serves as a visual guide. (d) Near-UV CD spectra of myristoylated Rc and RE demonstrate a decrease in signal intensity at 280 nm when calcium is added (+m, –Ca²⁺ → +Ca²⁺), reflecting a reduction of asymmetry in the environment of the tryptophan residues. This suggests that comparable tertiary structural changes occur in Rc and in the R-domain of the fusion.

Figure 3

Myristoylation- and calcium-dependent variations in the temperature-responsive phase behavior of RE fusions. (a,b) Variable-temperature turbidimetry shows the effects of myristoylation (±m) and calcium (±Ca²⁺) on the phase behavior of both constituent domains and RE fusions. (a) The R-control is thermally stable up to 55 °C, while the E-control exhibits a distinct, calcium-independent phase transition. (b) Fusion constructs display unique behaviors, including single or dual phase transitions, modulated by myristoylation and calcium. Error bars indicate standard deviations from duplicate measurements. (c–f) Thermal shift assays monitor temperature-triggered changes in the R-domain of the chimera using SYPRO Orange dye, which preferentially binds to exposed hydrophobic regions of the R-domain. (c) In the absence of myristoylation and calcium (−m, −Ca²⁺), (d) with myristoylation alone (+m, −Ca²⁺), (e) with calcium alone (−m, +Ca²⁺), and (f) with both myristoylation and calcium (+m, +Ca²⁺). Myristoylation and particularly calcium binding increase the melting temperature of the R-domain. With both modifiers present, the RE chimera exhibits two distinct thermal transitions at 37 and 66 °C, attributed to different structural changes within the R-domain: the lower transition suggests an emergent structural rearrangement, while the higher temperature corresponds to the melting point of the R-domain. Dashed lines and shaded areas indicate the standard deviation from three measurements. The red line in each panel represents the first derivative of the averaged curve.

Figure 4

Emergent nanoassembly of RE fusions is influenced by the interaction between myristoylation (±m) and calcium (±Ca²⁺). (a) Variable-temperature dynamic light scattering (DLS) elucidates that nanoassembly of RE fusions is regulated by both myristoylation and calcium, and their interaction is statistically significant above the transition temperature (T_E). Error bars represent standard deviations from two independent samples, each measured in triplicate. In each temperature regime, compact display letters are used for multiple comparisons; groups that do not share the same letter differ significantly, as determined by the Tukey test at the 5% significance level. Statistical significance of factors is indicated by p-values: ns (p > 0.5), * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001). For detailed ANOVA results, see Table S4. (b,c) Transformed DLS results highlight the temperature-dependent interactions of myristoylation and calcium in the R-control (b) and RE chimera (c). The DLS data confirm that the liposwitching thresholds align closely with TE and TR (indicated by vertical dashed lines), supporting the hypothesis that calcium-dependent exposure of the myristoyl group modulates E-domain aggregation. (d) Modifying the composition of the E-domain by increasing its hydrophilicity (E′) and decreasing its length (E″) can be used to modulate the liposwitching threshold.

Figure 5

Nanoassembly of RE fusion constructs. (a–d) Cryo-TEM at 35 °C visualizes the nanoscale organization of RE assemblies under different conditions: (a) without myristoylation or calcium (−m, −Ca²⁺); (b) with myristoylation alone (+m, −Ca²⁺); (c) with calcium alone (−m, +Ca²⁺); and (d) with both myristoylation and calcium (+m, +Ca²⁺). (e–g) Confocal microscopy is used to visualize the mesoscale organization of RE assemblies at 40–50 °C: (e) without myristoylation or calcium (−m, −Ca²⁺); (f) with myristoylation alone (+m, −Ca²⁺); and (g) with both myristoylation and calcium (+m, +Ca²⁺).