Quantitative analysis of FRET assay in biology —New developments in protein interaction affinity and protease kinetics determinations in the SUMOylation cascade

Abstract

Förster resonance energy transfer (FRET) techniques have been widely used in biological studies in vitro and in 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, K_d, and protease kinetics parameters, k_cat and K_m, in the SUMOylation pathway. The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.

Key words： quantitative FRET analysis; protein affinity determination; kinetics analysis

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