Fluorescence fluctuation spectroscopy on viral-like particles reveals variable gag stoichiometry

Area of Science:

• Biophysics
• Molecular Biology
• Virology

Background:

• Fluorescence fluctuation spectroscopy (FFS) quantifies particle properties like brightness, size, and concentration.
• Brightness analysis in FFS measures fluorescently labeled proteins, previously used for small oligomer stoichiometry.
• This study extends FFS brightness analysis to large macromolecular protein complexes.

Purpose of the Study:

• To determine the stoichiometry of human immunodeficiency virus type 1 (HIV-1) Gag protein within viral-like particles (VLPs).
• To investigate if fluorescent labeling of Gag protein affects VLP stoichiometry and size.
• To assess the relationship between Gag stoichiometry, VLP size, and Gag protein distribution.

Main Methods:

• Utilized fluorescence fluctuation spectroscopy (FFS) to analyze brightness of fluorescently labeled HIV-1 Gag protein in VLPs.
• Performed control experiments to confirm labeling does not influence VLP stoichiometry or size.
• Quantified Gag stoichiometry by correlating particle brightness with the amount of labeled Gag within VLPs.

Main Results:

• VLP stoichiometry and size were unaffected by fluorescent labeling of HIV-1 Gag protein.
• Particle brightness scaled linearly with the amount of labeled Gag protein incorporated into VLPs.
• HIV-1 Gag stoichiometry in VLPs varied significantly (approx. 750–2500 Gag proteins) with DNA plasmid amount, while VLP size remained constant.
• Observed that large areas of the VLP membrane are not covered by Gag protein.

Conclusions:

• HIV-1 Gag stoichiometry within VLPs is not constant and depends on Gag protein expression levels.
• A closed layer of Gag protein at the membrane is not essential for VLP formation.
• FFS brightness analysis is a valuable tool for quantitative analysis of large molecular complex composition and size.