Bottom-up assembly of hydrogels from bacteriophage and Au nanoparticles: the effect of cis- and trans-acting factors

Abstract

Hydrogels have become a promising research focus because of their potential for biomedical application. Here we explore the long-range, electrostatic interactions by following the effect of trans-acting (pH) and cis-acting factors (peptide mutation) on the formation of Au-phage hydrogels. These bioinorganic hydrogels can be generated from the bottom-up assembly of Au nanoparticles (Au NP) with either native or mutant bacteriophage (phage) through electrostatic interaction of the phage pVIII major capsid proteins (pVIII). The cis-acting factor consists of a peptide extension displayed on the pVIII that mutates the phage. Our results show that pH can dictate the direct-assembly and stability of Au-phage hydrogels in spite of the differences between the native and the mutant pVIII. The first step in characterizing the interactions of Au NP with phage was to generate a molecular model that identified the charge distribution and structure of the native and mutant pVIII. This model indicated that the mutant peptide extension carried a higher positive charge relative to the native pVIII at all pHs. Next, by monitoring the Au-phage interaction by means of optical microscopy, elastic light scattering, fractal dimension analysis as well as Uv-vis and surface plasmon resonance spectroscopy, we show that the positive charge of the mutant peptide extension favors the opposite charge affinity between the phage and Au NP as the pH is decreased. These results show the versatility of this assembly method, where the stability of these hydrogels can be achieved by either adjusting the pH or by changing the composition of the phage pVIII without the need of phage display libraries.

Figure 1. Scheme of Au-phage hydrogel assembly with native and mutant phage.

The pVIII and pIII arrows point to the major (white region) and minor phage (red region) capsids, respectively (not drawn to scale).

Figure 2. Calculations for native and mutant pVIII charge.

(A) Theoretical calculation based on individual amino acid charge of the native and mutant pVIII protein as a function of pH. (B) pVIII structure generated using DeepView images of the native (top) and a mutant version (bottom) of the pVIII protein. The positively charged domains are shown in blue and negative ones are in red. Hydrophobic regions appear white. The native sequence was: NH₃ ⁺-aegddpakaafdslqasateyigyawam vvvivgatigiklfkkfts-COO⁻ and the mutant one was NH₃ ⁺-PRQIKIWFQNRRMKWKKPae gddpakaafdslqasateyigyawamvvvivgatigiklfkkfts-COO⁻ (capitalized residues and boxed region represent the mutant peptide extension).

Figure 3. Effect of pH in the formation and structure of hydrogels assembled with native or mutant phage.

(A) Vials with hydrogels prepared with native and mutant phage at pH 2.0 (top) and 5.5 (bottom). Au NP of 50±8 nm were prepared by citrate reduction. (B) Darkfield micrographs (elastic scattering) of hydrogels in (A) (constant light intensity). (Scale bar, 10 µm). (C) Df of structures in (B) determined with box counting Df analysis (ImageJ software).

Figure 4. Differences in light extinction and SPR signal between the native and mutant Au-phage hydrogels as a function of pH.

(A) Light extinction at 750 nm for hydrogels as a function of pH. (B) SPR measurements of phage interaction with citrate-treated Au surface vs. pH (buffer background signal subtracted from data). (C) Surface charge density calculation based on individual amino acid charge present on mutant peptide extension.