Figure 2. Strategy to study dynamics of dimeric FoxP1 at the single-molecule level.
a) Cartoon representation summarizing the ten pseudohomodimers generated with single-cysteine mutants, showing the positions of the fluorescence acceptors (red) and donors (green). It is worth noting that acceptors and donors are located in different polypeptide chains. The secondary structure elements across the amino acid chain are shown to clarify the orientation of the single-cysteine mutants. b) Time-resolved fluorescence for pseudohomodimers of FoxP1 at single–molecule level was used to identify the limiting states. For all samples, two models, one or two lifetimes in addition to No FRET signal, was fitted. Residuals for the first and the second model are shown in olive and orange, respectively (Materials and Methods). c) MFD plots showing the distribution of events with specified ⟨τD(A)⟩f and FD/FA for dimers SS and VV. The black and red lines indicate the static and dynamic FRET lines, respectively. Orange and purple lines indicate the DS and the dimeric intermediate ensemble ({I2(k)}) states described using the lifetime measurements. FD/FA values above the gray line represents states beyond the distance range practically resolvable by FRET. d) The relative population of each FRET state for each pseudohomodimer derived with PDA analysis. e) Correlation plot comparing modeled DS interdye distances with the FRET derived distances obtained by smMFD. f) Kinetic model generated from time-resolved decays and PDA analysis, where the expected DS is in equilibrium with a heterogenous dimeric intermediate ensemble ({I2(k)}). The presence of static -the unsolved and fast kinetic exchange- (represented as circular arrows) and the characterized dynamic exchange in the model represents the complexity of the reaction.