High-pressure studies reveal the conformational landscape of the small GTPase Arf6 and other related studies

Despite significant sequence and structural homology in both the GDP- and GTP-bound forms to the more well-characterized protein Arf1, Arf6 exhibits distinct spatial and functional segregation: Arf1 mediates membrane trafficking between the Endoplasmic Reticulum and Golgi Apparatus, while Arf6 is responsible for trafficking between the plasma membrane and endosome. Furthermore, the accessory proteins required for in vivo functionality of the switch are not interchangeable. Arf proteins undergo large structural rearrangements during the nucleotide switch transition, especially in the N-terminal and switch regions, suggesting that partner specificity does not stem purely from end-state structures. We hypothesize that excited conformations sampled during this transition are responsible for the observed specificity. To characterize these excited conformational states, we utilize pressure-induced perturbation techniques that locally unfold the protein. Using a two-pronged approach of high-pressure NMR and high-pressure SAXS, we developed residue-specific unfolding curves for Arf6 and pair-distance distribution curves to understand the global shape of the protein at various pressures. These results enable structural mapping of the conformational landscape of Arf6.