Complex Analysis of Fluorescence Intensity Fluctuations of Molecular Compounds
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Journal of Applied Spectroscopy, Vol. 87, No. 4, September, 2020 (Russian Original Vol. 87, No. 4, July–August, 2020)
COMPLEX ANALYSIS OF FLUORESCENCE INTENSITY FLUCTUATIONS OF MOLECULAR COMPOUNDS M. M. Yatskou,a* V. V. Skakun,a L. Nederveen-Schippers,b A. Kortholt,b and V. V. Apanasovichc
UDC 535.37:547.96
A method is proposed for the complex analysis of fluctuations in the fluorescence intensity of molecular compounds, which allows determining the structural composition of protein oligomers. The idea of the method is to analyze the photon counting histograms of experimental measurements using principal component analysis to assess the presence of oligomeric compounds, and to perform hierarchical cluster analysis, to determine the data classes corresponding to various molecular compounds, followed by selecting cluster medoids to determine the oligomeric composition of protein complexes. The efficiency of the analysis algorithms developed within the framework of the proposed method was confirmed on simulated and experimental photon counting histograms of the measured fluorescence intensity fluctuations of monomeric and dimeric forms of green-fluorescent protein (GFP). Keywords: fluorescence intensity fluctuation, photon counting histogram, molecular compounds, protein oligomers, data mining, principal component analysis, hierarchical cluster analysis, green-fluorescent protein (GFP). Introduction. Fluorescence fluctuation spectroscopy is widely used to study the diffusion of proteins and their interactions in living cells [1–3]. In the course of the experiment, the fluorescence of molecules bound or freely moving in a solution or a cell is recorded in a certain small volume (up to 10–18 m3) formed by an extremely focused laser beam. Fluctuations in fluorescence intensity are primarily due to changes in the number and location of molecules in the recorded volume, as well as their interaction and the properties of the medium. The oligomeric composition of a protein compound can be determined by analyzing the amplitude of fluctuations in fluorescence intensity over time (methods for analyzing the distribution of fluorescence intensity — PCH (photon counting histogram) [4] and FIDA (fluorescence intensity distribution analysis) [5]). In the PCH and FIDA methods, a histogram of the number of photocounts (PC) is plotted at a given recording time interval to determine the concentration of a protein freely emitting or labeled with a luminescent dye. The recorded fluorescence intensity of the sample is directly proportional to the number of fluorescent molecules that form the studied molecular complex, which makes it possible to estimate the number of molecules inside the protein complex and the size of the complex [6, 7]. To analyze the distribution of the number of photocounts, various mathematical models [4–7] and optimization methods are usually used, among which the least squares method with Levenberg–Marquardt optimization [8] is used most often, which makes it possible to obtain information on the diffusion and st
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