Work Performed:
To attain our objectives, we first engineered protein constructs allowing to analyze, using Optical Tweezers, the coupled folding and binding of NCBD with its interaction partners—so we designed and produced constructs of NCBD fused with its corresponding binding partner through a polypeptide linker. Next, we optimized protocols to perform measurements on our fusions using Optical Tweezers by attaching the protein constructs to microspheres via DNA handles. Finally, we also developed specific methods to analyze and interpret the wealth of data obtained from different types of Optical Tweezers measurements.
Main Results:
In constant velocity measurements, the coupled folding and binding of NCBD with ACTR or SRC-1 is observed as a clear transition in Force-extension traces. In contrast with typical folding/unfolding transitions of structured proteins—more frequently detected as discrete jumps—for those protein fusions, coupled folding and binding transitions are observed as a highly dynamic exchange between a high-force state (associated with the fully bound and folded complex) and a low-force state (associated with the unbound and unfolded interactors) in the low picoNewtons. Conversely, for the fusion of NCBD with p53TAD no clear transition is observed, but coupled folding and binding could still be perceived as a subtle kink in Force-extension traces also in the low picoNewtons. The difference observed, depending on the specific interactor, is consistent with the known lower affinity of p53TAD to bind NCBD.
We could also record the kinetics of coupled folding and binding of NCBD with either ACTR or SRC-1 in constant distance measurements for long periods of time—for several minutes up to an hour. The resulting traces revealed the coupled folding and binding of NCBD with either ACTR or SRC 1 is characterized by very fast transitions between apparently only two states: the fully bound and folded (high force) and the completely unbound and unfolded (low force). However, a thorough analysis of kinetic data shows the disorder to order transitions of these protein complexes cannot be explained by a simple two state model—but requires to consider at least two kinetic regimes, involving the population of additional states. This finding is in agreement with previous results from our group using smFRET . More relevantly, our experiments using Optical Tweezers revealed a completely novel feature of the coupled folding and binding of NCBD with either ACTR or SRC-1 where the interaction partners dwell in an unbound and unfolded, apparently non productive or “locked”, state for tens of milliseconds to several seconds.
Given the role of Proline isomerization in the kinetic behavior previously evidenced with smFRET, we designed a collection of Proline mutants to survey the effect of their isomerization on the kinetic features observed under force . As in smFRET experiments, a specific Proline seems to mainly govern the switching between kinetic regimes—but using Optical Tweezers we find that its mutation does not fully prevent the flux between the two regimes, suggesting a minor involvement of other Prolines. Interestingly, the absence of Prolines does not preclude the population of the “locked” state; but some Prolines seem to influence the frequency and lifetime of its population.
Since Proline isomerization alone could not explain the population of this new “locked” state, we evaluated (focusing on the NCBD-ACTR model) whether it could result from specific properties of the single interacting partners—so we studied NCBD and ACTR alone using Optical Tweezers. Interestingly, while ACTR does not present any detectable transition in constant velocity measurements, NCBD shows a subtle but clearly recognizable transition in Force extension traces. We could also record kinetics of the intrinsic transitions of NCBD in constant distance measurements, and we observe that NCBD populates a lower force state with a lifetime in a range of few seconds. We hypothesize these transitions may correspond to the full unfolding of the Molten Globule conformation NCBD is considered to populate in isolation, and that disruption of this Molten Globule may explain the population of the “locked” state by the complex fusions.
