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

Risako Kobayashi,Hiroshi Inaba,Kazunori Matsuura

doi:10.3390/ijms22094754

Risako Kobayashi, Hiroshi Inaba + Show 1 more

Open Access

https://doi.org/10.3390/ijms22094754

Copy DOI

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.

Highlights

The interior of living cells is often referred to as the ‘molecular crowding environment’, in which many biomacromolecules, such as proteins, nucleic acids, and carbohydrates, exist in high density [1]
We report in situ fluorescence correlation spectroscopy (FCS) analysis of artificial viral capsids formed by the co-assembly of fluorescent BODIPY-labelled and unmodified β-annulus peptides in dilute aqueous solutions and under molecular crowding conditions
The peptide was purified by reversed-phase high-performance liquid chromatography (HPLC), and its structure was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) (m/z = 2408 [M]+, Figure S1)

Summary

Introduction

The interior of living cells is often referred to as the ‘molecular crowding environment’, in which many biomacromolecules, such as proteins, nucleic acids, and carbohydrates, exist in high density [1]. Over the past two decades, it has been revealed that the structure, activity, and stability of biomacromolecules in molecular crowding environments, which mimic the cellular environment, considerably differ from those in dilute aqueous solutions. To elucidate the self-assembling process of natural viral capsids, intermediate structures have been previously detected by ion mobility separation-mass spectrometry, interferometric scattering microscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM) under dilute conditions [14,15,16]. The formation of viral capsids by self-assembly occurs in molecular crowding environments, such as a host cell [17]. Mateu and co-workers reported that macromolecular crowding led to the reversible assembly of CA, human immunodeficiency virus type 1 capsid protein, into capsid-like particles at low ionic strength, which was different from diluted conditions [18]

Methods

Results

Conclusion