Single-Molecule FRET Reveals the Native-State Dynamics of the IκBα Ankyrin Repeat Domain

Abstract

Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1–4), at ARs 2 and 5 (AR 2–5), and at ARs 3 and 6 (AR 3–6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 “loses grip”. AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.

A Disorder-Induced Domino-Like Destabilization Mechanism Governs the Folding and Functional Dynamics of the Repeat Protein IκBα

“…For example, for donor/acceptor probes located at AR 1 and AR 4 and at 298 K, a broad peak centered at high FRET values is seen (blue in Figure 3D) indicating that the protein is well-folded though with fluctuations from sampling partially-structured conformations (I 2 ; Figure 3A). At 310 K, the predicted mean FRET is lower and broader as a result to populating more disordered regions as represented by increasing distances for lower values of RC, exactly as observed in experiments [15]. Though there is a correlation between experiments and simulations on the mean FRET values of the first and second peak (Figure 3H) we do not capture the experimental amplitude, i.e.…”

Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα

“…By placing the donor and acceptor fluorophores at various positions, Lamboy et al (14) were able to obtain information about the flexibility of each repeat with respect to others, and the results revealed that AR1 fluctuates in both the free and NFkB-bound states, whereas AR2 and AR4 never appear to fluctuate. Fluctuations were observed when FRET pairs were placed at AR3 and AR6, but since fluctuations were also observed when the FRET pairs were placed at AR2 and AR6, these fluctuations were attributed to fluctuation of AR6 (14).…”

Probing protein disorder and complexity at single-molecule resolution

“…A later smFRET study by the same authors showed that the AR 6 domain was more prone to disorder than the AR 5 domain. Thus, while the AR 5 domain did not show disorder at room temperature (requiring an increase to 37 °C to show disorder), the AR 6 domain demonstrated disordered even at room temperature 29 .…”