Novel Quantitative FRET Assays for the Sumoylation Pathway Analysis

Förster resonance energy transfer (FRET) is a widely used method that determines molecular distance of 1-10nm which fits well with biomolecular interactions and conformational changes. FRET has been widely used in biological and biomedical research in vitro and in vivo. We recently developed a novel quantitative FRET analysis methodology and applied this method to determine protein interaction affinities and protease kinetics in the SUMOylation cascade. The novel methodology is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor emission, acceptor emission and FRET signal. SUMOylation is an important protein post-translational modification, which is carried out by multi-step enzymatic cascade reactions for peptide activation and substrate conjugation, and plays critical roles in diverse physiological processes, including transcriptional regulation, signal transduction, cell survival and death and DNA damage response. We developed a new theoretical and experimental procedure for protein interaction K_d determination of SUMO1 and its E2 ligase, Ubc9, and individual interaction in the full SUMOylation cascade by FRET assay. The K_d values (~0.3mM) are in good agreement with those determined with other traditional approaches, such as surface plasmon resonance(SPR) (0.35mM) and isothermal titration calorimetry(ITC)(0.25mM). We have applied the same strategy to develop a novel quantitative FRET-based protease assay for SENP kinetics parameter determination. SENP is a family of proteases that are responsible for SUMO maturation from precursor and removal from conjugated substrates. The novel theoretical and experimental procedures to determine the kinetics parameters, _kcat, K_M and catalytic efficiency (k_cat/K_M = 2.49 x 10⁷ M^-1s^-1) that is superior than traditional biochemical assays. We have applied this novel methodology for K_d determinations of entire SUMOylation cascade and SENP proteases. These developments represent a novel methodology of biosensor based on FRET for systems kinetics analysis, which can be used in other biosystems and applied for drug discoveries.