ESI-IM-MS and Collision-Induced Unfolding That Provide Insight into the Linkage-Dependent Interfacial Interactions of Covalently Linked Diubiquitin.

Abstract

Understanding protein higher order structure and interfacial interactions is crucial to understanding protein binding motifs and cellular function, that is, an interactome. Polyubiquitylation is a post-translational modification that functions as a tag for a diverse array of cellular processes, wherein differences in chain length, branching, and linkage site encode different cellular functions. Investigation of covalently linked diubiquitin (diUbq) molecules specifically selects for the effect of covalent linkage site on the conformational preference of the molecule and the interfacial interactions between the subunits. Here, we report results for electrospray ionization ion mobility-mass spectrometry (ESI-IM-MS) and collision-induced unfolding (CIU) analysis of four diUbq ions to provide new understanding of the differences in subunit interfacial interactions and conformational preferences induced by the four most common covalent linkage sites. The specific hydrophobic patch interface adopted by K48-linked diUbq results in unique CIU fingerprints dominated by conformational broadening and primarily gradual unfolding, as opposed to the distinct transitions through gas-phase unfolding intermediates observed of K6-, K11-, and K63-linked diUbq. Comparison of the intermediate conformational families of K6-, K11-, and K63-linked diUbq suggests that K6- and K11-linked diUbq adopt a mixture of conformers stabilized by either electrostatic interactions or hydrophobic interactions involving the I36 hydrophobic patch. Furthermore, conditions favoring the partially folded A-state of monoubiquitin, that is, methanolic solution, induce conformational collapse and distinct unfolding intermediates for all four linkage types, providing an end-point at which all solution-phase conformational "memory" has been lost.