RfaH is a compact two‐domain bacterial transcription factor that functions both as a regulator of transcription and an enhancer of translation. Underpinning the dual functional roles of RfaH is a partial but dramatic fold switch, which completely transforms the ~50‐amino acid C‐terminal domain (CTD) from an all‐α state to an all‐β state. The fold switch of the CTD occurs when RfaH binds to RNA polymerase (RNAP), however, the details of how this structural transformation is triggered is not well understood. Here we use all‐atom Monte Carlo simulations to characterize structural fluctuations and mechanical stability properties of the full‐length RfaH and the CTD as an isolated fragment. In agreement with experiments, we find that interdomain contacts are crucial for maintaining a stable, all‐α CTD in free RfaH. To probe mechanical properties, we use pulling simulations to measure the work required to inflict local deformations at different positions along the chain. The resulting mechanical stability profile reveals that free RfaH can be divided into a “rigid” part and a “soft” part, with a boundary that nearly coincides with the boundary between the two domains. We discuss the potential role of this feature for how fold switching may be triggered by interaction with RNAP.
We consider a generalized-ensemble algorithm for coarse-grained simulations of biomolecules which allows the thermodynamic behavior of two or more sequences to be determined in a single multisequence run. By carrying out a random walk in sequence space, the method also enhances conformational sampling. Escape from local energy minima is accelerated by visiting sequences for which the minima are more shallow or absent. We test the method on an intermediate-resolution coarse-grained model for protein folding with 3 amino acid types and explore the potential for a large-scale coverage of sequence space by applying the method to sets of more than 1000 sequences. The resulting thermodynamic data are used to analyze the structures and stability properties of sequences covering the space between folds with different secondary structures.
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