Abstract
Protein–protein interactions and enzyme-catalyzed reactions are the fundamental processes in life, and the quantification and manipulation, kinetics determination, and ether activation or inhibition of these processes are critical for fully understanding physiological processes and discovering new medicine. Various methodologies and technologies have been developed to determine the parameters of these biological and medical processes. However, due to the extreme complexity of these processes, current methods and technologies can only determine one or a few parameters. The recent development of quantitative Förster resonance energy transfer (qFRET) methodology combined with technology aims to establish a high-throughput assay platform to determine protein interaction affinity, enzymatic kinetics, high-throughput screening, and pharmacological parameters using one assay platform. The FRET assay is widely used in biological and biomedical research in vitro and in vivo and provides high-sensitivity measurement in real time. Extensive efforts have been made to develop the FRET assay into a quantitative assay to determine protein–protein interaction affinity and enzymatic kinetics in the past. However, the progress has been challenging due to complicated FRET signal analysis and translational hurdles. The recent qFRET analysis utilizes cross-wavelength correlation coefficiency to dissect the sensitized FRET signal from the total fluorescence signal, which then is used for various biochemical and pharmacological parameter determination, such as KD, Kcat, KM, Ki, IC50, and product inhibition kinetics parameters. The qFRET-based biochemical and pharmacological parameter assays and qFRET-based screenings are conducted in 384-well plates in a high-throughput assay mode. Therefore, the qFRET assay platform can provide a universal high-throughput assay platform for future large-scale protein characterizations and therapeutics development.Graphical
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