Fluorescence Correlation Spectroscopy Analysis of Effect of Molecular Crowding on Self-Assembly of β-Annulus Peptide into Artificial Viral Capsid - PubMed (original) (raw)

Fluorescence Correlation Spectroscopy Analysis of Effect of Molecular Crowding on Self-Assembly of _β_-Annulus Peptide into Artificial Viral Capsid

Risako Kobayashi et al. Int J Mol Sci. 2021.

Abstract

Recent progress in the de novo design of self-assembling peptides has enabled the construction of peptide-based viral capsids. Previously, we demonstrated that 24-mer _β_-annulus peptides from tomato bushy stunt virus spontaneously self-assemble into an artificial viral capsid. Here we propose to use the artificial viral capsid through the self-assembly of _β_-annulus peptide as a simple model to analyze the effect of molecular crowding environment on the formation process of viral capsid. Artificial viral capsids formed by co-assembly of fluorescent-labelled and unmodified _β_-annulus peptides in dilute aqueous solutions and under molecular crowding conditions were analyzed using fluorescence correlation spectroscopy (FCS). The apparent particle size and the dissociation constant (Kd) of the assemblies decreased with increasing concentration of the molecular crowding agent, i.e., polyethylene glycol (PEG). This is the first successful in situ analysis of self-assembling process of artificial viral capsid under molecular crowding conditions.

Keywords: artificial viral capsid; fluorescence correlation spectroscopy; molecular crowding; self-assembly; β-annulus peptide.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

(A) Schematic illustration of the construction of a fluorescent-labelled artificial viral capsid by co-assembly of BODIPY-_β_-annulus and _β_-annulus peptides. (B,C) Measured (solid) and fitted (dot) autocorrelation curves for a 0.1 μM BODIPY-_β_-annulus peptide (green) and a mixture of 0.1 μM BODIPY-_β_-annulus peptide and 200 μM _β_-annulus peptide (red) measured by FCS in 10 mM Tris-HCl buffer (pH 7.0) at 25 °C. The lower graph shows the residual plot. (D) Normalized autocorrelation curves of a mixture of 0.1 μM BODIPY-_β_-annulus and _β_-annulus peptides at 0~200 μM in 10 mM Tris-HCl buffer (pH 7.0) at 25 °C.

Figure 2

Figure 2

(A,B) Concentration dependence of _β_-annulus peptides on the apparent diameter (A) and ratio (B) of the fast component (green) to the slow component (red) determined by FCS curve fitting (N = 3) at a constant concentration of the BODIPY-_β_-annulus peptide (0.1 μM in 10 mM Tris-HCl buffer) (pH 7.0) at 25 °C. (C) Hill plot of binding of the _β_-annulus peptides to the BODIPY-_β_-annulus peptides in a 10 mM Tris-HCl buffer (pH 7.0). The fractions obtained from FCS analysis were fitted into the Hill equation to determine the apparent dissociation constant Kd.

Figure 3

Figure 3

(A) Schematic illustration of the construction of fluorescent-labelled artificial viral capsids by co-assembly of BODIPY-_β_-annulus and _β_-annulus peptides in the presence of molecular crowding agents. (B,C) Measured (solid) and fitted (dot) autocorrelation curves for 0.1 μM BODIPY-_β_-annulus peptides (purple) and a mixture of 0.1 μM BODIPY-_β_-annulus and 200 μM _β_-annulus peptides (pink) measured by FCS in the presence of 5 wt% polyethylene glycol (PEG2000) in a 10 mM Tris-HCl buffer (pH 7.0) at 25 °C. The bottom graph shows residual plots. (D) Normalized autocorrelation curves of a mixture of 0.1 μM BODIPY-_β_-annulus and _β_-annulus peptides at 0–200 μM in the presence of 5 wt% PEG2000 in a 10 mM Tris-HCl buffer (pH 7.0) at 25 °C.

Figure 4

Figure 4

Concentration dependence of _β_-annulus peptides on the ratio of the fast (green) and slow components (red) determined by FCS curve fitting (N = 3) at a constant concentration of the BODIPY-_β_-annulus peptide (0.1 μM) in the presence of 5 wt % (A), 10 wt% (B), 15 wt% (C), 20 wt% (D) PEG2000 at 25 °C.

Figure 5

Figure 5

Effect of PEG2000 on the apparent diameter (A) and ratio (B) of the fast (green) and slow components (red) determined by FCS curve fitting (N = 3) in the presence of PEG2000 at constant concentrations of the BODIPY-_β_-annulus (0.1 μM) and _β_-annulus peptides (25 μM) at 25 °C.

Similar articles

Cited by

References

    1. Fulton A.B. How crowded is the cytoplasm? Cell. 1982;30:345–347. doi: 10.1016/0092-8674(82)90231-8. - DOI - PubMed
    1. Nakano S., Miyoshi D., Sugimoto N. Effects of molecular crowding on the structures, interactions, and functions of nucleic acids. Chem. Rev. 2014;114:2733–2758. doi: 10.1021/cr400113m. - DOI - PubMed
    1. Minton A.P. Implications of macromolecular crowding for protein assembly. Curr. Opin. Struct. Biol. 2000;10:34–39. doi: 10.1016/S0959-440X(99)00045-7. - DOI - PubMed
    1. Minton A.P. The Influence of Macromolecular Crowding and macromolecular confinement on biochemical reactions in physiological media. J. Biol. Chem. 2001;276:10577–10580. doi: 10.1074/jbc.R100005200. - DOI - PubMed
    1. Ma Q., Fan J.-B., Zhou Z., Zhou B.-R., Meng S.-R., Hu J.-Y., Chen J., Liang Y. The contrasting effect of macromolecular crowding on amyloid fibril formation. PLoS ONE. 2012;7:e36288. doi: 10.1371/journal.pone.0036288. - DOI - PMC - PubMed

MeSH terms

Substances

LinkOut - more resources