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Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli.

Hur KH, Mueller JD - PLoS ONE (2015)

Bottom Line: Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes.We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements.The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

View Article: PubMed Central - PubMed

Affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
The brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined it with the analysis of axial scans through the E. coli cell. The MSQ method recovers brightness, concentration, and diffusion time of soluble proteins in E. coli. We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements. We further used MSQ analysis to determine the oligomeric state of nuclear transport factor 2 labeled with EGFP expressed in E. coli cells. The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

No MeSH data available.


Related in: MedlinePlus

Ratio of gamma factors.The ratio γ2,cyl (R, 0)/γ2,∞ as determined from Eq 19 is shown as a function of the radius R. The dashed line indicates the values for the measured E. coli cells.
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pone.0130063.g007: Ratio of gamma factors.The ratio γ2,cyl (R, 0)/γ2,∞ as determined from Eq 19 is shown as a function of the radius R. The dashed line indicates the values for the measured E. coli cells.

Mentions: The ratio γ2,cyl (R, 0)/γ2,∞ calculated from Eq 19 is shown in Fig 7 as a function of the radius R. Because the radius of the E. coli bacteria was constant at 0.45 μm, the gamma ratio for E. coli is γ2,cyl (0.45 μm, 0)/γ2,∞ = 1.51.


Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli.

Hur KH, Mueller JD - PLoS ONE (2015)

Ratio of gamma factors.The ratio γ2,cyl (R, 0)/γ2,∞ as determined from Eq 19 is shown as a function of the radius R. The dashed line indicates the values for the measured E. coli cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476568&req=5

pone.0130063.g007: Ratio of gamma factors.The ratio γ2,cyl (R, 0)/γ2,∞ as determined from Eq 19 is shown as a function of the radius R. The dashed line indicates the values for the measured E. coli cells.
Mentions: The ratio γ2,cyl (R, 0)/γ2,∞ calculated from Eq 19 is shown in Fig 7 as a function of the radius R. Because the radius of the E. coli bacteria was constant at 0.45 μm, the gamma ratio for E. coli is γ2,cyl (0.45 μm, 0)/γ2,∞ = 1.51.

Bottom Line: Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes.We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements.The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

View Article: PubMed Central - PubMed

Affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACT
The brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined it with the analysis of axial scans through the E. coli cell. The MSQ method recovers brightness, concentration, and diffusion time of soluble proteins in E. coli. We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements. We further used MSQ analysis to determine the oligomeric state of nuclear transport factor 2 labeled with EGFP expressed in E. coli cells. The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

No MeSH data available.


Related in: MedlinePlus