Solution structure of lysine‐free (K0) ubiquitin

Lysine‐free ubiquitin (K0‐Ub) is commonly used to study the ubiquitin‐signaling pathway, where it is assumed to have the same structure and function as wild‐type ubiquitin (wt‐Ub). However, the K0‐Ub ¹⁵N heteronuclear single quantum correlation NMR spectrum differs significantly from wt‐Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt‐Ub. The three‐dimensional structure of K0‐Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0‐Ub adopts the same backbone structure as wt‐Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations.