Predicting the loading of virus-like particles with fluorescent proteins

Area of Science:

• Biotechnology
• Biophysics
• Materials Science

Background:

• Virus-like particles (VLPs) from Cowpea Chlorotic Mottle Virus (CCMV) are versatile platforms for encapsulating foreign molecules.
• Controlling the loading of cargo within VLPs is crucial for applications in drug delivery and nanobiotechnology.

Purpose of the Study:

• To investigate rational design approaches for loading teal fluorescent proteins (TFP) into CCMV VLPs.
• To analyze the impact of controlled cargo loading on molecular confinement effects and protein interactions within the VLP.

Main Methods:

• Development of covalent and noncovalent strategies for TFP encapsulation into CCMV VLPs.
• Utilizing fluorescence anisotropy to measure homo-Förster Resonance Energy Transfer (homo-FRET) and assess protein interactions.
• Characterization of VLP loading efficiency and prediction of cargo capacity.

Main Results:

• Covalent loading enabled precise control over TFP encapsulation at a molecular level.
• Loading of over 10 TFP molecules into the 18 nm CCMV capsid cavity induced maximum homo-FRET efficiency.
• Molecular crowding effects were observed to influence protein behavior even at low VLP loading levels.

Conclusions:

• Rational covalent design offers precise control over VLP cargo loading and facilitates prediction of encapsulation.
• Molecular confinement within CCMV VLPs significantly impacts the efficiency of homo-FRET between encapsulated proteins.
• This study highlights the role of molecular crowding in VLP-based systems.