Intrinsically disordered proteins (IDPs) lack stable 3-dimensional structure by themselves but can fold when bound to a partner protein. Hif-1α is a transcription factor involved in regulating the cellular response to hypoxia that possesses a disordered C-terminal transactivation domain (CTAD). CTAD folds into a three helical structure when bound to the Taz1 domain of the CREB-binding protein (CBP), a transcriptional coactivator. Here, we use 3-color single-molecule FRET to dissect the mechanism of coupled folding/binding of Hif-1α CTAD to CBP Taz1. Site specific labeling of Hif-1α with donor (D) and acceptor 1 (A1) dyes at the respective N and C termini and Taz1 with an acceptor 2 (A2) dye at either terminus allows for monitoring of multiple distances. All dwell time distributions fit well to a single exponential, implying a single rate-limiting kinetic event in both binding and dissociation. Kinetic data from binding and dissociation of Taz1 to immobilized Hif-1α exhibit slow dissociation dynamics, with respective association rate constants of 7.2 x 10 6 and 4.8 x 10 6 M -1 s -1 for N- and C-terminus labeled Taz1 and respective dissociation rate constants of 0.76 and 0.68 s -1 . Dissociation constants for binding of N- and C-terminus labeled Taz1 were 100 and 140 nM, respectively. Transition density plots reveal distinct clusters corresponding to transitions between the 3-color bound, 2-color bound (A2 on bound Taz1 absent/photobleached), and unbound states, recapitulating the dwell time analysis.
Carney et al. (Sun,) studied this question.