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Protein phase separation plays a very important role in many biological processes and is closely related to the occurrence and development of some serious diseases. So far, the fluorescence imaging method and fluorescence correlation spectroscopy (FCS) have been frequently used to study the phase separation behavior of proteins. Due to the wide size distribution of protein condensates in phase separation from nano-scale to micro-scale in solution and living cells, it is difficult for the fluorescence imaging method and conventional FCS to fully reflect the real state of protein phase separation in the solution due to the low spatio-temporal resolution of the conventional fluorescence imaging method and the limited detection area of FCS. Here, we proposed a novel method for studying the protein phase separation process by objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS). In this study, CRDBP proteins were used as a model and respectively fused with fluorescent proteins (EGFP and mCherry). We first compared conventional FCS and Scan-FCS methods for characterizing the CRDBP protein phase separation behaviors and found that the reproducibility of Scan-FCS is significantly improved by the scanning mode. We studied the self-fusion process of mCherry-CRDBP and EGFP-CRDBP and observed that the phase change concentration of CRDBP was 25 nM and the fusion of mCherry-CRDBP and EGFP-CRDBP at 500 nM was completed within 70 min. We studied the effects of salt concentration and molecular crowding agents on the phase separation of CRDBP and found that salt can prevent the self-fusion of CRDBP and molecular crowding agents can improve the self-fusion of CRDBP. Furthermore, we found the recruitment behavior of CRDBP to β-catenin proteins and studied their recruitment dynamics. Compared to conventional FCS, Scan-FCCS can significantly improve the reproducibility of measurements due to the dramatic increase of detection zone, and more importantly, this method can provide information about self-fusion and recruitment dynamics in protein phase separation. [ABSTRACT FROM AUTHOR]

The identification of molecular targets for achieving beneficial effects from small-molecule drugs is a crucial and currently unsolved challenge, which leads to high costs and long development cycles. Therefore, it is urgent to develop methods for easily and quickly acquiring information about protein–drug interaction at a molecular level. In this study, we propose a novel method for the study of protein–drug interaction by fluorescence correlation spectroscopy (FCS) based on organic solvent-induced protein aggregation. We used β-secretase (BACE-1) and dihydrofolate reductase (DHFR) as model proteins. Fluorescence-labelled proteins aggregated in aqueous solutions containing organic solvents. In the presence of drugs, the aggregation of proteins was inhibited greatly, and FCS was used to characterize protein aggregates. The decrease in the characteristic diffusion time (τD) of protein aggregates demonstrated a strong interaction between proteins and drug molecules. We presented a new parameter IC50 to assess the inhibitory effects of drugs on the basis of the changes in the τD of fluorescence-labelled proteins under different concentrations of the drugs in the presence of organic solvents. We acquired a remarkable difference in the IC50 values for different drugs and in terms of the trend, our results were consistent with those reported by other methods. Compared with current methods, our approach is simple, low-cost, and time-saving, and has the potential to become a promising and universal tool for drug screening at the molecular level. [ABSTRACT FROM AUTHOR]