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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

Experimental z-scan intensity profiles of EGFP from E. coli cell.Experimental z-scan intensity data (diamonds) from eight consecutive z-scans together with model function (red curve) for F∞= 72 kcps and R = 0.45 μm.
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pone.0130063.g006: Experimental z-scan intensity profiles of EGFP from E. coli cell.Experimental z-scan intensity data (diamonds) from eight consecutive z-scans together with model function (red curve) for F∞= 72 kcps and R = 0.45 μm.

Mentions: We performed eight consecutive z-scans through the geometric center of an E. coli cell with reduced laser power to ensure the absence of photobleaching during the scans. The intensity profiles of the consecutive scans are shown in Fig 6. Each profile was fit by Eq 18 to determine F∞ and R, which recovered the averaged fit parameters F∞ = 72±2 kcps and R = 0.45±0.01 μm. Inserting the averaged fit parameters into Eq 18 resulted in a modeled intensity profile (red solid line, Fig 6), which is in good agreement with the experimental data. We repeated this experiment on several E. coli cells (n = 14). The peak intensity differed for each cell, reflecting the variations in the EGFP concentration from cell to cell. However, the radius was essentially identical for all cells. The averaged radius was 0.45±0.026 μm.


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

Hur KH, Mueller JD - PLoS ONE (2015)

Experimental z-scan intensity profiles of EGFP from E. coli cell.Experimental z-scan intensity data (diamonds) from eight consecutive z-scans together with model function (red curve) for F∞= 72 kcps and R = 0.45 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130063.g006: Experimental z-scan intensity profiles of EGFP from E. coli cell.Experimental z-scan intensity data (diamonds) from eight consecutive z-scans together with model function (red curve) for F∞= 72 kcps and R = 0.45 μm.
Mentions: We performed eight consecutive z-scans through the geometric center of an E. coli cell with reduced laser power to ensure the absence of photobleaching during the scans. The intensity profiles of the consecutive scans are shown in Fig 6. Each profile was fit by Eq 18 to determine F∞ and R, which recovered the averaged fit parameters F∞ = 72±2 kcps and R = 0.45±0.01 μm. Inserting the averaged fit parameters into Eq 18 resulted in a modeled intensity profile (red solid line, Fig 6), which is in good agreement with the experimental data. We repeated this experiment on several E. coli cells (n = 14). The peak intensity differed for each cell, reflecting the variations in the EGFP concentration from cell to cell. However, the radius was essentially identical for all cells. The averaged radius was 0.45±0.026 μm.

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