FCAT: Fluorescence Correlation Analysis Tool

FCAT is designed for the analysis of protein fluorescence spectra by revealing the spectral components of individual tryptophan residues, or groups of tryptophan residues located close to each other and having energy transfer among them. As a result of the FCAT module calculations, the fluorescence spectra will be decomposed and spectral components will be assigned to one of the five spectral-structural classes.

Input of data

There are 2 steps in input of spectral data.

Step 1. FCAT could be used for the decomposition of single spectrum or set of fluorescence spectra (not less than 3) measured at increasing concentration of quencher of tryptophan fluorescence such as Acrylamide ; Cs+; I- or NO3-.
In case of decomposition of single tryptophan spectrum press “One”
In case of decomposition of set of fluorescence spectra, press “More than one”
Step 2. is designed to choose a way to submit the spectral data. The first option is to upload the input file. The other option is to fill out the web-forms and the input file would be generated automatically, and it would be displayed in the result window.

In case of single spectrum the input file should contain 4 lines:

Line 1
Name of protein, conditions, comments, etc. The information is processed as a whole line.

Line 2
This line must contain 3 numbers, which are:

Line 3
Contains the spectrum (fluorescence intensities)

Line 4
The fluorescence spectrum of tryptophan residue in solution (recorded at the same setting of instrument as used for the measurement of protein spectrum, but with Lex = 280 nm). It would be used for the calculation of the correction curve.

In all lines except the first one, values must be separated by spaces or commas.
The example of input file could be found on the web-site

 

In case of set of spectra the input file should contain the following lines:

Line 1
Name of protein, conditions, comments, etc. The information is processed as a whole line.

Line 2
This line must contain 5 numbers, which are:

Line 3
Enumerate the quencher concentrations (in M) in increasing order; the first concentration must be zero.

Next Lines
Contain the spectra (fluorescence intensities) at increasing quencher concentrations (each line should present one spectrum).

Final Line
The fluorescence spectrum of tryptophan residue in solution (recorded at the same setting of instrument as used for the measurement of protein spectra, but with Lex = 280 nm). It would be used for the calculation of the correction curve.

In all lines except the first one, values must be separated by spaces or commas.
The example of input file could be found on the web-site

Tips: protein fluorescence measurements

  • The best range for the measurements of tryptophan fluorescence spectra is from 310 to 400 nm. The decomposition calculations will be performed in range of 320-380 nm to exclude the contribution of light scattering at short wavelengths and reduce errors of low intensities at long wavelengths.

  • The step of wavelengths at which the spectral points are recorded should be not less than 1 nm and not exceeding of 5 nm (there is no significant improvement of the decomposition results at step of less than 1 nm, while the time of calculations increase dramatically).

  • The decomposition analysis is designed for the analysis of tryptophan fluorescence, therefore the excitation wavelength should be > 295 nm (in case if protein contains tyrosine residues) and could be 280 nm (if there are no tyrosine residues in protein).

  • The best way is to keep the excitation and emission slits at 2 nm. However, if the signal is not strong enough, first, increase the excitation slit and then emission slit

  • Each spectrum should be recorded not longer than 1-2 minutes to avoid the decrease of intensity at long wavelengths due to the tryptophan photobleaching (the slow kinetics measurements of intensity changes at any wavelength (340 nm for example) could be used to monitor photobleaching at the experimental set up used for spectra recording).

  • Avoid using of sodium azide (NaN3)

  • Avoid using of samples of high turbidity. Use front face configuration of cuvette chamber, where the light is collected from the front face rather than at 90C as is on majority of spectrofluorometers.

  • Keep temperature as a constant during the fluorescence measurements. The fluorescence signal depends on temperature!

  • To reduce Wood’s anomalies from reflecting holographic grating, the polarizers in the excitation and emission paths should be set at “magic” angle (54.7 °from the vertical orientation) and vertically (0°), respectively.

  • The concentrated solution of quencher used in fluorescence measurements should be prepared fresh just before the experiment.

  • The quencher concentrations used in experiment is recommended not to exceed 0.4 M in cuvette.

  •  Minimum 3 spectra should be recorded for the decomposition analysis. The optimal number of quencher concentrations (in other words, numbers of fluorescence spectra) is 5-7.

  • The addition of quencher in cuvette must decrease (or does not change) the fluorescence intensity. If the result is opposite, the experiment should be redone!

  • In case of ionic quenchers such as KI, CsCl or NaNO3 need to keep the total ionic strength constant by addition of KCl (for example, if you plan to measure protein fluorescence spectra at 4 various quencher concentrations: 0, 0.05, 0.1, 0.15 and 0.2 M, you need to add KCl in each sample in concentration of 0.2, 0.15, 0.1, 0.005 and 0 M to keep the total ionic strength in solution at 0.2 M).

  • In case of non-ionic quencher, acrylamide, it might be added directly into cuvette. In that case the dilution factor should be taken into account.

  • The fluorescence spectrum of an aqueous solution of L-tryptophan must be included in input file. The tryptophan spectrum should be measured at the same experimental set up as measurements of protein spectra. The tryptophan spectrum is used for the calculation of correction for the instrument spectral sensitivity. The concentration of tryptophan should be the same, which gives the absorbance signal OD=0.2-0.3.

FCAT calculations

The decomposition of tryptophan fluorescence spectra is expected to be completed in 1-20 minutes (depending on the number of spectra under the treatment the step and some other factors). If the result will not appear in 30 minutes, please contact PFAST administrator.

Output file: Summary.txt

The results of fluorescence decomposition analysis are presented in the form of three ASCII files (results, graph-data, summary) and one graph file. The Summary.txt file contains summary of the analysis.

Line 1
Name of protein, conditions, comments, etc. It is first line of input file

Line 2
Date of calculation

Table
The table contains the summarized information of the decomposition analysis:

  • The algorithm used for the analysis (detailed information about each algorithm could be found in Background Section and papers). Note: PHREQ can decompose only into two components! PHREQ data would be presented only in case of more than one spectrum used in the analysis (the spectra measured at difference concentrations of quencher). In case of decomposition of single spectrum, the PHREQ results do not exist.
  • DISCR. - goodnes-of-Fit. (average  error of decomposition). NOTE: The output files contain information about the decomposition analysis into one, two and three components performed separately. The user has to choose which solution to take as final result. The DISCR. Should be used as a criterion:  the lower the value of the DISCR, the better is solution!
  • Nsc - number of smoothed cycles used during the calculations to eliminate noise.
  • N - number of spectral components
  • Lm, nm - the positions of maxima of the spectral components in wavelengths with values of the standard deviations
  • S, % -the fluorescence contribution of the spectral component to the total spectrum in percents with the values of the standard deviations.
  • CLASS - the assignment of the spectral components to the one of five spectral-structural classes. Note: Information about spectral –structural classes could be found in Background section.

Next Lines
Notes about the parameters presented in file

Output file: Result.txt

The results of fluorescence decomposition analysis are presented in the form of three ASCII files (results, graph-data, summary) and one graph file. The Result.txt file contains detailed informatio of the analysis.

Line 1
Name of protein, conditions, comments, etc. It is first line of input file

Line 2
Date of calculation

Line 3-4
Concentration and recalculated activity values of quencher. Note: The values of concentration and activity would differ only in case of ionic quenchers. In case of acrylamide, the values would be the same.

The results of analysis by applying of various methods (PHREQ and SIMS) are presented separately in tables. Note: PHREQ can decompose only into two components! PHREQ data would be presented only in case of more than one spectrum used in the analysis (the spectra measured at difference concentrations of quencher). In case of decomposition of single spectrum, the PHREQ results do not exist.

Table 1

The table 1 contains values of the maximum position of spectral components in wavelengths (L, nm) and wavenumbers (V, 1/cm) and the standard deviations values (DL and DV). Note: PHREQ can decompose only into two components!

Table 2

The table 2 contains i) contributions of spectral components into the area under the spectrum in % (Si, %)(with standard deviations, s.d., %)  and ii) the values of the area under spectral components (Ai) calculated for the spectra measured at various quencher concentration (Cq, M). Last column presents the values of area under the whole spectrum (Atotal=Sum(Ai))

Table 3

The table 3 contains

  1. Ksv, 1/M - Stern-Volmer quenching constant (in M-1) and standard deviations, s.d., %
  2. Ksv/Ksv(TRP), %  - relative quenching constant (100% corresponds to the Stern-Volmer constant of Trp in solution, Ksv(Trp)) and standard deviations, s.d., %)
  3. Bi - free term in Stern-Volmer equation. Note: It should be equal 1, the larger is deviation of this value from 1 the worse is solution (see equation 2 in Background Section)
  4. R - linear correlation coefficients for Stern-Volmer plots (goodness of fit of Stern-Volmer plot)

Last Line for each subprogram
DISCR. - goodnes-of-fit the same value, which is presented in Summary.txt file


Output file: Graph Data.txt

The Graph Data.txt file is generated to arrange all input and output values in a convenient way for the plotting of graphs.

Line 1
Name of protein, conditions, comments, etc. It is first line of input file

Line 2
Date of calculation

Next Lines contains original input or normalizes and corrected input data
The values of experimental spectra (data taken from the input file)

Correction curve calculated by using of the inputted tryptophan spectrum and the tryptophan spectrum used as a standard (it is included in programs of calculations). All experimental spectra would be multiplied by the correction curve.
 
The values of normalized corrected experimental spectra

Next Lines contains the results of analysis of each algorithm presented separately
Note: PHREQ can decompose only into two components! PHREQ data would be presented only in case of more than one spectrum used in the analysis (the spectra measured at difference concentrations of quencher). In case of decomposition of single spectrum, the PHREQ results do not exist.

The values of smoothed normalized corrected experimental spectra used in the decomposition analysis

Differences between experimental and smoothed normalized corrected experimental spectra at each wavelength

Lines: Data for the Stern-Volmer plot


Line 1: Quencher chemical activities, M

Next Line(s): Experimental points for the Stern-Volmer plot for each spectral component

Next Line(s): Theoretical values for the Stern-Volmer plot for each spectral component

Note:

  1. In case of single-component solution (SIMS-I), it would be 1 line with experimental points and 1 line with theoretical values of Stern-Volmer plot
  2. In case of two-components solution (SIMS-II or PHREQ), it would be 2 lines with experimental points for the Stern-Volmer plot for each spectral component and 2 lines with theoretical values for the Stern-Volmer plot for each spectral component
  3. In case of three-components solution (SIMS-III), it would be 3 lines with experimental points for the Stern-Volmer plot for each spectral component and 3 lines with theoretical values for the Stern-Volmer plot for each spectral component.

Lines: Residuals, %
Difference between theoretical and experimental spectra.

Lines: Theoretical spectra from 305 to 400, Step 1nm
Note: It would be as many theoretical spectra as many spectra were included in analysis.

Lines: Spectral components for the first theoretical spectrum (spectrum with 0 concentration of quencher) from 305 to 400, Step 1nm
Note: In case of single-component solution (SIMS-I) it would be 1 spectrum
In case of two-components solution (SIMS-II or PHREQ) it would be 2 spectra.
In case of three-components solution (SIMS-III) it would be 3 spectra

Last Line for each subprogram
DISCR. - goodnes-of-fit the same value, which is presented in Summary.txt and Results.txt files.


Visualize Graph

It provides already plotted results, which contains:

Residuals, %, It is difference between theoretical and experimental spectra

Experimental points of the first spectrum (measured at 0 concentration of quencher) (black dots) and calculated spectral components: component 1 (red line), component 2 (green line) and component 3 (blue line), the sum of components presented as black line.

The Stern-Volmer plots constructed for each spectra component.