• Categories:FCS Technology for Molecular Interaction Analysis
• Time of issue:2023-05-24 14:41
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(Summary description)The team of Kazuhiro Aoki of the National Institute of Natural Sciences of Japan applied fluorescence correlation spectroscopy ( FCS , FCCS ) to quantitatively analyze the concentration and dissociation constant of endogenous protein molecules in a single living cell. Traditional in vitro biochemical experiments cannot reflect the true level of intracellular protein concentration and interaction, and fluorescent labeling of endogenous proteins is very difficult. In view of these research difficulties, the author first fluorescently labeled the endogenous proteins ERK2 and RSK2 in Hela cells by CRISPR/Cas9 technology , and measured ERK2-mEGFP and RSK2-HaloTag-TMR in a single Hela cell using FCS and FCCS technology Concentrations and dissociation constants of two endogenous fusion proteins. It is known that both FBS and EGF can stimulate the nuclear translocation of ERK2 in the cytoplasm . In order to verify the validity of the expression of endogenous fusion protein, the author used the method of fluorescence microscopy to observe the expression of ERK2-mEGFP and RSK2 in Hela cells stimulated by FBS or EGF . -The distribution change of HaloTag-TMR ; In addition, the author used FCS technology to determine the measurement noise in the cell and confirmed that the variability of protein concentration in the cell was greater than that between cells, in order to illustrate the endogenous fusion protein concentration, dissociation constant measurement Data Validity. The protein concentration and dissociation constant in 198 Hela cells were measured in the experiment , and the average concentrations of ERK2-mEGFP and RSK2-HaloTag-TMR were obtained by autocorrelation analysis as 0.078 μM and 0.097 μM , and the ERK2 and RSK2 in a Hela cell were estimated The total concentrations were 0.68 μ M and 0.50 μ M ; the average K d value in vivo between ERK2-mEGFP and RSK2-HaloTag-TMR was 0.59 μ M through cross-correlation analysis . In order to further study the signal transduction mechanism of endogenous proteins after EGF stimulated cells, the author used FCS and FCCS to measure cytoplasmicThe concentration and dissociation constant of the two endogenous fusion proteins in the / nucleus vary with time. The results show that ERK2-mEGFP transfers from the cytoplasm to the nucleus after EGF stimulation, and the K d value in the cytoplasm increases instantaneously 10 minutes after EGF stimulation . The complex is more stable in the nucleus. This article establishes a method for the quantitative determination of endogenous protein concentration and molecular interaction dissociation constant in living cells using CRISPR/Cas9 genome editing technology and FCS/FCCS, which solves the difficulty of fluorescent labeling of endogenous proteins in living cells, and in the living cell environment . It is difficult to analyze the molecular mechanism in situ; compared with the traditional exogenous protein fluorescent labeling method, this method can truly reflect the parameters such as protein concentration in the cell, and provide accurate input parameters for the dynamic modeling of the molecular mechanism of living cells. FCCS is a technique that can be used to analyze molecular interactions in situ in living cells, and is of great significance in the field of drug development.