Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across two subdomains of a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the C-terminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization and not specific tertiary interactions.
Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-function in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. Surface plasmon resonance experiments found that Hsp90
Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the Cterminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization.Keywords: protein folding, effective concentration, cooperativity, helix stabilization, T4 lysozyme, In this work, we investigate how a helix spanning the two subdomains of T4 lysozyme* couples distant regions of the protein. We find evidence for a model of long-distance coupling that relies on the cooperative nature of helix formation to stabilize a long-range tertiary salt bridge interaction at one end of the helix and thereby couple the folding of T4 lysozyme's subdomains. This mechanistic model may have implications for co-translational folding. IntroductionCooperativity is a hallmark of globular proteins. At equilibrium, many small (<200 residues) globular proteins fold in an apparent two-state manner (U⇌N), populating either a completely folded or unfolded conformation 1,2 . The stability of the folded protein (ΔG UN = G N -G U ) is the sum of many interactions, both local and long-range. While the chemical nature and mechanism of the short-range, local interactions can be easy to identify and investigate, it is difficult to probe the long-range interactions that cause the structure and energetics in one region to be coupled to those in another region and thus lead to two-state behavior. For repeat proteins, such as ankrin domains, this coupling has been attributed to a large interfacial energy that, in some cases, overshadows the lack of intrinsic stability of the individual repeats 3,4 .Here, we explore coupling between distant regions of a protein using the protein T4 lysozyme*, T4L* (* denotes the cysteine-free pseudo-wild type variant 5 ). T4L* is a well-studied globular protein; the stabilities and structures of hundreds of...
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