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 simulate the folding and fold switching of the C-terminal domain (CTD) of the transcription factor RfaH using an all-atom physics-based model augmented with a dual-basin structure-based potential energy term. We show that this hybrid model captures the essential thermodynamic behavior of this metamorphic domain, that is, a change in the global free energy minimum from an α-helical hairpin to a 5-stranded β-barrel upon the dissociation of the CTD from the rest of the protein. Using Monte Carlo sampling techniques, we then analyze the energy landscape of the CTD in terms of progress variables for folding toward the two folds. We find that, below the folding transition, the energy landscape is characterized by a single, dominant funnel to the native β-barrel structure. The absence of a deep funnel to the α-helical hairpin state reflects a negligible population of this fold for the isolated CTD. We observe, however, a higher α-helix structure content in the unfolded state compared to results from a similar but fold switch-incompetent version of our model. Moreover, in folding simulations started from an extended chain conformation we find transiently formed α-helical structure, occurring early in the process and disappearing as the chain progresses toward the thermally stable β-barrel state. | INTRODUCTIONProteins are increasingly being discovered with a remarkable ability to switch between folds with widely different structures. [1][2][3] While it is not uncommon for proteins to undergo large-scale motions after their initial folding, such as domain-swapping [4] or other hinge-like motions, [5] fold switching is a distinct phenomenon. It involves a reorganization of the protein at the most basic structural level at play in folding, that is, secondary structure (α-helices and β-sheets). Despite the remarkable complexity of these molecular transformations, fold switching is reversible and thereby controlled by the system's free energy. In this sense, it can be said that metamorphic proteins adhere to Anfinsen's thermodynamic principle (or hypothesis) of protein folding. [6] Clearly, however, fold switching fundamentally challenges the idea of a unique native conformation, which was a central aspect of the classic view of folding since emerging from the pioneering refolding experiments on ribonuclease A. [7] It is important to note that fold switching typically occurs only when triggered by specific changes to the local environment (or milieu) of the protein, such as salt concentration, [8] redox condition [9] or oligomerization state. [10] In the absence of such a trigger, metamorphic proteins typically fold to an apparently unique structure, which masks their fold switching capabilities. As a result, metamorphic proteins often go unrecognized. [11] Metamorphic proteins thus encode two different folds within a single amino acid sequence even though, as mentioned, they typically adopt a single fold for a given (constant) local milieu. It is natural, then, to ask: what impact does this dual encoding have on their fol...

We study the folding and fold switching of the C-terminal domain (CTD) of the transcription factor RfaH using a hybrid sequence-structure based model. We show that this model captures the essential thermodynamic behavior of this metamorphic domain, i.e., a switch in the global free energy minimum from an α-helical hairpin to a 5-stranded β-barrel upon separating the CTD from the rest of the protein. Using this model and Monte Carlo sampling techniques, we analyze the energy landscape of the CTD in terms of progress variables for folding towards the two folds. We find that, below the folding temperature, the energy landscape is characterized by a single, dominant funnel to the native β-barrel structure. The absence of a deep funnel to the α-helical hairpin state reflects a negligible population of this fold for the isolated CTD. We observe, however, a significantly higher α-helix structure content in the unfolded state compared to results from a similar but fold switch-incompetent version of our model. Moreover, in folding simulations started from an extended chain conformation we find transient α-helix structure that disappears as the chain progresses to the thermally stable β-barrel state.

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