Extended Data Fig. 2: Single-molecule FRET analysis of wild-type human DHX36 and bovine DHX36 constructs.
From: Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36
a, Schematic of the smFRET assay22,28. See Extended Data Fig. 8 for FRET state assignments. b, Binding of wild-type human DHX36 (DHX36-WT)22 to the G-quadruplex substrate, induces a shift from a high to medium and low FRET states (grey and cyan histograms, respectively). The shift is interpreted as the binding of DHX36 to the G-quadruplex substrate. Upon buffer flow, dissociation is not observed (purple histogram). Wild-type human DHX36 displays repetitive unfolding activity22, as indicated by the oscillation between medium and low FRET states after binding to the G-quadruplex substrate (blue trace). c, Binding of wild-type bovine DHX36 (incorporating a KKK192AAA mutation to prevent spontaneous proteolysis; DHX36-AAA) to the G-quadruplex substrate induces a shift from a high FRET state to medium and low FRET states (grey and cyan histograms, respectively). The shift is interpreted as the binding of DHX36 to the G-quadruplex substrate. Upon buffer flow, dissociation is not observed (purple histogram). Wild-type bovine DHX36 (DHX36-AAA) displays repetitive unfolding activity, as indicated by the oscillation between low and medium FRET states after binding to the G-quadruplex substrate (blue trace). FRET traces are shown for two molecules. d, Deletion of residues 111–159, mutation EEK435YYY, and mutation KDTK752AATA to generate DHX36-DSM does not impair G-quadruplex binding or repetitive unfolding activity. FRET traces are shown for two molecules. e, Dwell time comparison between human DHX36-WT (grey bars), bovine wild-type DHX36 (DHX36-AAA, cyan bars) and bovine DHX36-DSM (orange bars). All three proteins show a comparable FRET range, and the two bovine constructs exhibit similar dwell times between the medium and low FRET states. Dwell times between the bovine constructs and the human construct are different, probably owing to interspecies differences. Each experiment was performed three times. Data are reported as box dot plots, with the data centre as the median ± s.e. of 1,000 dwell times from 200 representative molecules. f–h, Mutation of motif IVa (hook loop) (f), the OB subdomain residue R856 (g), and OII does not result in impaired repetitive unfolding activity (h). However, partial dissociation following washing is observed with the motif IVa (f) and OII mutation (h). i, Pre-incubation of bovine DHX36-AAA with the non-hydrolysable ATP γ-phosphate hydrolysis transition state mimic ADP•AlF4− does not affect repetitive unfolding activity on G-quadruplex substrates. j, Addition of ATP (red arrow) while DHX36-AAA is displaying repetitive unfolding activity on G-quadruplex substrates results in DHX36 dissociation (blue arrow) on the seconds timescale. Each experiment was repeated three times with highly similar results. Each measurement yields data from at least 10,000 molecules.
