Conformational changes induced in ubiquitin by circular protein-DNA chimeras
TL;DR
The NMR-scale synthesis of circular chimeras of single-and double-stranded DNA with ubiquitin, an essential component of many cellular pathways, is described and the results establish both the promise of this strategy and the need to account for local DNA-protein interactions.
Abstract
Proteins are dynamic molecular machines that change shape in response to physical and chemical perturbations. Although single-molecule force spectroscopy provides precise information about the stretching of proteins in response to tunable forces, it does so without structural detail. Circular protein-DNA chimeras, with DNA attached to pairs of surface sites, have been introduced as an alternative way to tunably apply forces to proteins. Intriguingly, these chimeras should be tractable for atomic-level study by nuclear magnetic resonance (NMR) spectroscopy and other structural methods. Here, we describe the NMR-scale synthesis of circular chimeras of single-and double-stranded DNA with ubiquitin, an essential component of many cellular pathways. We designed these chimeras to probe a two-residue retraction of ubiquitin’s C-terminal β5 strand, normally triggered by phosphorylation of serine 65 during initiation of mitophagy. We probed the resulting conformational changes by NMR and found that the attachment of a single strand of DNA suffices to alter this conformational equilibrium. A control bearing two separate short single DNA strands recapitulated much of the circular chimera’s NMR properties, supporting a dominant role for local protein-DNA interactions rather than spring-like action by single- or double-stranded DNA. These results provide a necessary benchmark for future studies using DNA springs to probe the functional dynamics of proteins. Significance statement Ligands, post-translational modifications, and mechanical inputs reshape proteins through forces that propagate across their structures. The underlying mechanical properties of proteins mediating these changes are rarely accessible with atomic-level detail. By producing NMR-scale circular protein-DNA chimeras, we provide a route to examining how defined physical perturbations alter protein conformational land-scapes. The results establish both the promise of this strategy and the need to account for local DNA-protein interactions.