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

Abstract

Fluorescence fluctuation spectroscopy determines the brightness, size, and concentration of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used previously to determine the stoichiometry of small oligomers in cells. We extend brightness analysis to large macromolecular protein complexes containing thousands of proteins and determine their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to determine the stoichiometry of HIV-1 Gag within the particles. Control experiments establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The experiments further show that the brightness scales linearly with the amount of labeled Gag within the particle. Brightness analysis shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not constant, but varies with the amount of transfected DNA plasmid. We observed HIV-1 Gag stoichiometries ranging from approximately 750 to approximately 2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP production. This study shows that brightness analysis has the potential to become an important tool for investigating large molecular complexes by providing quantitative information about their size and composition.

Figure 1

FFS data for a VLP sample. (A) Fluorescence intensity time trace. (B) PCH data (diamonds) and fit (solid line) to a two species model. The first species is very dim with a brightness equal to that of a control sample void of VLPs. The second, bright species has a normalized brightness of ∼750. (C) A fit of the autocorrelation function determined a hydrodynamic diameter of 130 nm.

Figure 2

Power dependence of a VLP sample. (A) The concentration is shown as a function of excitation power. PCH analysis determined the average particle number N in the observation volume, which was converted into a concentration by dividing by the observation volume. (B) The brightness determined by PCH analysis is shown as a function of excitation power together with a fit to a power law. The fitted exponent of 1.98 agrees within error with the expected quadratic power-law dependence.

Figure 3

Positively-stained transmission electron micrograph of VLP samples. Two Gag VLP particles are shown on top of the figure, whereas the bottom depicts two Gag^YFP VLP particles. Image analysis of 66 stained Gag VLP particles determined an average diameter of 129 ± 12 nm. Analysis of 33 Gag^YFP VLP particles identified an average diameter of 130 ± 17 nm.

Figure 4

Stoichiometry of Gag^YFP in the presence of unlabeled Gag. VLPs produced from cells cotransfected with Gag^YFP and Gag plasmid. The Gag^YFP copy number is shown as a function of mole fraction of labeled Gag plasmid. The graph shows two sets of independent measurements. A linear fit to the function b(y) = b(1) y with b(1) = 750 is shown as a solid line.

Figure 5

SDS-PAGE gel (12.5%) of VLP samples containing Gag^YFP and Gag. (A) The mole fraction of labeled Gag plasmid is indicated at the bottom of each lane. The bands are fluorescently stained with SYPRO ruby protein gel stain and imaged. Two bands with positions that agree with the molecular weights of Gag and Gag^YFP are present (bottom band: Gag; top band: Gag^YFP; middle band: BSA from the cell medium). (B) Mole fraction of Gag^YFP in VLPs containing a mixture of Gag^YFP and Gag, as established by quantitation of bands on a denaturing polyacrylamide gel. The graph displays the mole fraction of Gag^YFP protein as a function of the mole fraction of labeled Gag plasmid used for cell transfection. The line through the origin with a slope of one illustrates the linear relationship between the protein mole fraction and the plasmid mole fraction.

Figure 6

(Left) About 5000 copies of Gag protein are required to cover the surface area of a 140-nm particle. (Right) Our experimental data indicate that 750 copies of Gag protein are sufficient to form viral like particles, which corresponds to a surface coverage of ∼15% as conceptually illustrated in the figure. For simplicity, evenly distributed Gag proteins are depicted. Recent data indicate that Gag proteins exist in patches (19).