Forster resonance energy transfer （FRET） techniques have been widely used in biological studies in vitro andin vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occur...Forster resonance energy transfer （FRET） techniques have been widely used in biological studies in vitro andin vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occurs between oscillating dipoles of two fluorophores with overlapping emission and excitation wavelengths and is dependent on the spectroscopic and geometric properties of the donor-acceptor pair. Various efforts have been made to develop quantitative FRET methods to accurately determine the interaction affinity and kinetics parameters. SUMOylation is an important post-translational protein modification with key roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases （SENP） act as endopeptidases to process the pre-SUMO or an isopeptidase to deconjugate SUMO from its substrate. Here we also summarize recent developments of theoretical and experimental procedures for determining the protein interaction dissociation constant, Kd, and protease kinetics parameters, kcat and Kin, in the SUMOylation pathway. The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.展开更多
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 ar...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 Forster 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 K_(D),K_(cat),K_(M),K_(i),IC_(50),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.展开更多
文摘Forster resonance energy transfer （FRET） techniques have been widely used in biological studies in vitro andin vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occurs between oscillating dipoles of two fluorophores with overlapping emission and excitation wavelengths and is dependent on the spectroscopic and geometric properties of the donor-acceptor pair. Various efforts have been made to develop quantitative FRET methods to accurately determine the interaction affinity and kinetics parameters. SUMOylation is an important post-translational protein modification with key roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases （SENP） act as endopeptidases to process the pre-SUMO or an isopeptidase to deconjugate SUMO from its substrate. Here we also summarize recent developments of theoretical and experimental procedures for determining the protein interaction dissociation constant, Kd, and protease kinetics parameters, kcat and Kin, in the SUMOylation pathway. The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.
基金supported by the National Institutes of Health Grant AI076504,the UCR Academic Senate Grant,and the Attaisina gift grant.
文摘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 Forster 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 K_(D),K_(cat),K_(M),K_(i),IC_(50),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.
