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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

Normalized brightness of EGFP from E. coli cells.MSQ-curves were fit to Eq 14 with A(Q1, 1) = Q1 and converted into a normalized brightness by b = Q1/Qcyl,∞. The normalized brightness is independent of the initial fluorescence intensity F0. The average brightness (dashed line) is 0.98 ± 0.09. The top axis represents the initial protein concentration, while the right axis displays the biased normalized brightness b* = Q1/QEGFP,∞, when the finite size of the bacterium is ignored.
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pone.0130063.g008: Normalized brightness of EGFP from E. coli cells.MSQ-curves were fit to Eq 14 with A(Q1, 1) = Q1 and converted into a normalized brightness by b = Q1/Qcyl,∞. The normalized brightness is independent of the initial fluorescence intensity F0. The average brightness (dashed line) is 0.98 ± 0.09. The top axis represents the initial protein concentration, while the right axis displays the biased normalized brightness b* = Q1/QEGFP,∞, when the finite size of the bacterium is ignored.

Mentions: Additional E. coli cells expressing EGFP were measured to test our analysis procedure. The FFS data taken at the geometric center were fit to Eq 14 and the normalized brightness was calculated from the recovered Q1 with the help of Eqs 4 and 20. The radius of the E. coli cell was either determined from the z-scan intensity profile or taken as 0.45 μm. We plotted the normalized brightness b versus the initial fluorescence intensity F0 (Fig 8). The values of b are close to 1 with a mean of 0.98 and a standard deviation of 0.09. This result correctly identifies the bacterially expressed EGFP as a monomeric protein. The right axis shows the biased normalized brightness b* = Q1/QEGFP,∞ that results if the incomplete PSF overlap is not accounted for. A value of b* close to 1.51 would lead to the misleading conclusion that the sample is a mixture of monomers and dimers. Thus, accounting for photodepletion and geometry of the bacterium is crucial to avoid misinterpretation of FFS brightness experiments inside E.coli.


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

Hur KH, Mueller JD - PLoS ONE (2015)

Normalized brightness of EGFP from E. coli cells.MSQ-curves were fit to Eq 14 with A(Q1, 1) = Q1 and converted into a normalized brightness by b = Q1/Qcyl,∞. The normalized brightness is independent of the initial fluorescence intensity F0. The average brightness (dashed line) is 0.98 ± 0.09. The top axis represents the initial protein concentration, while the right axis displays the biased normalized brightness b* = Q1/QEGFP,∞, when the finite size of the bacterium is ignored.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130063.g008: Normalized brightness of EGFP from E. coli cells.MSQ-curves were fit to Eq 14 with A(Q1, 1) = Q1 and converted into a normalized brightness by b = Q1/Qcyl,∞. The normalized brightness is independent of the initial fluorescence intensity F0. The average brightness (dashed line) is 0.98 ± 0.09. The top axis represents the initial protein concentration, while the right axis displays the biased normalized brightness b* = Q1/QEGFP,∞, when the finite size of the bacterium is ignored.
Mentions: Additional E. coli cells expressing EGFP were measured to test our analysis procedure. The FFS data taken at the geometric center were fit to Eq 14 and the normalized brightness was calculated from the recovered Q1 with the help of Eqs 4 and 20. The radius of the E. coli cell was either determined from the z-scan intensity profile or taken as 0.45 μm. We plotted the normalized brightness b versus the initial fluorescence intensity F0 (Fig 8). The values of b are close to 1 with a mean of 0.98 and a standard deviation of 0.09. This result correctly identifies the bacterially expressed EGFP as a monomeric protein. The right axis shows the biased normalized brightness b* = Q1/QEGFP,∞ that results if the incomplete PSF overlap is not accounted for. A value of b* close to 1.51 would lead to the misleading conclusion that the sample is a mixture of monomers and dimers. Thus, accounting for photodepletion and geometry of the bacterium is crucial to avoid misinterpretation of FFS brightness experiments inside E.coli.

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