Search Results (2)

Fluorescence fluctuation spectroscopy (FFS) techniques rely on determination of monomeric molecular brightness, i.e., the fluorescence intensity of a single, non-aggregated fluorophore, as a critical reference for estimating protein oligomer size. By comparing measured molecular brightness of fluorescently labeled proteins of interest to this monomeric brightness benchmark, FFS enables inference of oligomerization states. However, widely used fluorescent proteins often exhibit self-association, compromising monomeric brightness calibration and introducing errors in brightness-derived oligomer-size estimates. This study compares two strategies for determining monomeric brightness: the conventional fluctuation-based method and a more recently proposed average-intensity-based alternative. The comparison uses two model fluorophores, a fluorescent protein (mCitrine) and the small-molecule dye Janelia Fluor 525 (JF₅₂₅) conjugated to HaloTag. Our results show strong agreement between intensity- and fluctuation-derived brightness values only in the minimally aggregating JF₅₂₅–HaloTag benchmark. In contrast, in the mCitrine samples, where aggregation is more prevalent, only the intensity-based method maintains a consistent estimate across sample preparation, while the fluctuation-based method overestimates brightness when aggregation effects become pronounced. This robustness makes the intensity-based approach a valuable cross-check for monomeric brightness calibration. Our results support a combined strategy, using both methods to improve the reliability of monomeric brightness calibration and protein oligomerization analysis in FFS. Full article

Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500–2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag–Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag–Gag interactions that occur during virus particle assembly and in released immature particles. Full article