Modulating long‐range energetics via helix stabilization: A case study using T4 lysozyme

Abstract

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.

A saturation-mutagenesis analysis of the interplay between stability and activation in Ras

“…Both assays indicate that truncating the C-terminal helix destabilizes H-Ras by ~7 kJ⋅mol -1 . This effect is consistent with the known ability of terminal-helix stabilization to help maintain the entire protein fold (Rosemond et al, 2018).…”

Local Xenon–Protein Interaction Produces Global Conformational Change and Allosteric Inhibition in Lysozyme

“…Herein, we used bacteriophage T4L as a model system to study the impact of Xe on protein activity, both experimentally and computationally. T4L is a well-studied enzyme − that facilitates phage infection by breaking down the bacterial cell wall and promoting the release of virus . This is a concerted process where the peptide side chain is recognized by C-terminal helices, and the β-1,4 glycosidic bondbetween the repeating N-acetylmuramic (NAM) and N-acetylglucosamine (NAG) units that make up the cell wall peptidoglycanis hydrolyzed at the active site. , In the catalytic cycle, lysozyme smoothly and continuously changes between “open” and “closed” conformations: substrate binding induces a change from open to closed conformation to initiate hydrolysis of the glycosidic bond. − Once the bond is broken, the enzyme/product complex adopts a more compact conformation, and then the lysozyme “open form” is regenerated upon product release .…”