Observing Protein Interactions and Their Stoichiometry in Living Cells by Brightness Analysis of Fluorescence Fluctuation Experiments

Chen, Yan; Johnson, Jolene; Macdonald, Patrick J.; Wu, Bin; Mueller, Joachim D.

doi:10.1016/s0076-6879(10)72026-7

Cited by 50 publications

(60 citation statements)

References 35 publications

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“…The brightness doubling observed with EGFP 2 (Fig. 4) ensures that virtually all EGFP molecules of the cell-free system mature and acquire fluorescence [9]. The result further confirms the absence of long-lived dark states for the EGFP-fluorophore in two-photon excitation.…”

Section: Discussionsupporting

confidence: 54%

“…This, in turn, lowers the brightness from 2 to 1.7. A detailed discussion of the influence of dark states and flickering of fluorophores on brightness experiments is available [9]. The brightness of EGFP and EGFP 2 in mammalian cells is routinely measured as a control to establish that maturation and protein expression are not compromised.…”

Section: Discussionmentioning

confidence: 99%

“…However, there are a number of potential challenges for FFS studies in cell-free systems. The fidelity of cell-free expression and of chromophore maturation needs to be checked, because the presence of nonfluorescent or incompletely expressed protein leads to biased interpretation of FFS experiments [9]. We develop a droplet-based assay for FFS experiments in a cell-free extract.…”

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confidence: 99%

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Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Macdonald

Chen

Mueller

2012

Analytical Biochemistry

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Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.

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Section: Discussionsupporting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Macdonald

Chen

Mueller

2012

Analytical Biochemistry

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“…Fluorescence fluctuation spectroscopy (FFS) is a biophysical method for quantifying protein association directly inside the living cell 31 . The technique relies on a small optical observation volume created within the cell.…”

Section: Introductionmentioning

confidence: 99%

APOBEC3 Multimerization Correlates with HIV-1 Packaging and Restriction Activity in Living Cells

Chen

et al. 2014

Journal of Molecular Biology

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APOBEC3G belongs to a family of DNA cytosine deaminases that are involved in the restriction of a broad number of retroviruses including HIV-1. Prior studies have identified two distinct mechanistic steps in Vif-deficient HIV-1 restriction: packaging into virions and deaminating viral cDNA. APOBEC3A, for example, although highly active, is not packaged and is therefore not restrictive. APOBEC3G, on the other hand, although having weaker enzymatic activity, is packaged into virions and is strongly restrictive. Although a number of studies have described the propensity for APOBEC3 oligomerization, its relevance to HIV-1 restriction remains unclear. Here, we address this problem by examining APOBEC3 oligomerization in living cells using molecular brightness analysis. We find that APOBEC3G forms high-order multimers as a function of protein concentration. In contrast, APOBEC3A, APOBEC3C and APOBEC2 are monomers at all tested concentrations. Among other members of the APOBEC3 family, we show that the multimerization propensities of APOBEC3B, APOBEC3D, APOBEC3F, and APOBEC3H (haplotype II) bear more resemblance to APOBEC3G than to APOBEC3A/3C/2. Prior studies have shown that all of these multimerizing APOBEC3 proteins, but not the monomeric family members, have the capacity to package into HIV-1 particles and restrict viral infectivity. This correlation between oligomerization and restriction is further evidenced by two different APOBEC3G mutants, which are each compromised for multimerization, packaging, and HIV-1 restriction. Overall, our results imply that multimerization of APOBEC3 proteins may be related to the packaging mechanism and ultimately to virus restriction.

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“…In this study, we used a variety of fluorescence fluctuation spectroscopy (FFS) analysis techniques to examine the properties of EndoB1. Autocorrelation analysis (36) and brightness analysis (37) both revealed the existence of two distinct species of EndoB1, including a cytosolic, soluble fraction, and a much larger pool associated with small intracellular vesicles. Unlike our previous study that was limited to the detection of proteins associated with cellular vesicles, this work seeks to identify changes in the labeled vesicle population that occur as a result of a deliberate perturbation.…”

Section: Introductionmentioning

confidence: 99%

Association of Endophilin B1 with Cytoplasmic Vesicles

Baryłko

Mueller

et al. 2017

Biophysical Journal

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Endophilins are SH3-and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol. Here, we present results of fluorescence fluctuation spectroscopy analyses indicating that, in contrast, the majority of endophilin B1 is present in multiple copies on small, highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. Using dual-color heterospecies partition analysis, we identified the epidermal growth factor receptor on endophilin B1 vesicles. Moreover, a proportion of endophilin B1 vesicles also contained caveolin, whereas clathrin was almost undetectable on those vesicles. These results raise the possibility that endophilin B1 participates in dynamin 2-dependent formation of a population of transport vesicles distinct from those generated by A-type endophilins.

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Observing Protein Interactions and Their Stoichiometry in Living Cells by Brightness Analysis of Fluorescence Fluctuation Experiments

Cited by 50 publications

References 35 publications

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

APOBEC3 Multimerization Correlates with HIV-1 Packaging and Restriction Activity in Living Cells

Association of Endophilin B1 with Cytoplasmic Vesicles

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