# Improving the Thermostability of the Qβ Bacteriophage Coat Protein Through Single-Site Mutation Based on Molecular Dynamics

## TL;DR

This paper describes how to improve the stability of a virus-like particle using a single amino acid mutation, making it more reliable for virus detection.

## Contribution

The novel contribution is the use of molecular dynamics and rational design to create a more thermostable Qβ bacteriophage coat protein.

## Key findings

- Single-site mutations significantly improved the thermostability of Qβ virus-like particles.
- Mutant virus-like particles showed slower degradation rates compared to wild-type at various temperatures.
- The mutant particles were structurally intact and uniformly distributed.

## Abstract

Norovirus is a major cause of acute viral gastroenteritis in humans. Molecular biology-based detection methods play a pivotal role in ensuring accurate and specific diagnosis. The inclusion of Qβ phage particles as armored positive controls in these assays can further enhance their reliability and specificity. Herein, we discuss rational design strategies to improve the stability of Qβ bacteriophage capsid proteins armored with RNA using Discovery Studio 2019 protein design software. Amino acid mutation sites were deter-mined based on changes in folding free energy differences (ΔΔGmut). These single-site mutations were subsequently evaluated using molecular dynamics simulations. Wild-type and mutant recombinant expression plasmids were constructed and transformed into Escherichia coli BL21 (DE3) for cloning and expression. The stability of Qβ virus-like particles (VLPs) was assessed using real-time fluorescence RT-qPCR. The results showed that structurally intact and uniformly distributed wild-type and single-site mutant VLPs were successfully obtained. Stability analyses indicated that at 4 °C, 25 °C, 37 °C, 45 °C, and 60 °C, the single-site mutant exhibited a significantly lower rate of degradation than the wild-type. In conclusion, rational design enables the generation of single-site mutant VLPs with enhanced stability, providing a safer and more stable standard reference material for the molecular detection of foodborne viruses.