Scheme 1.

(a) Assembly of K11-linked Ub₂ from recombinant Ub monomers. The two Ubs are shaded differently to illustrate the concept of unit-specific isotopic labeling. In particular, the Ub₂ chain with an isotope-labeled proximal Ub (shaded gray) is assembled from the unlabeled Ub monomer and isotope-labeled K11Boc Ub. Similarly, Ub₂ with a labeled distal Ub is made from an isotope-labeled Ub and unlabeled K11Boc Ub. The same concept applies to making Ub₂ chains linked via other lysines (by incorporating Lys(Boc) at other Lys positions) and/or with both Ub units labeled but with different isotopes (²H, ¹⁵N, ¹³C or combination thereof) uniformly or using different residue- or group-specific labeling schemes. (b) Structure of Alloc and Boc protecting groups on a lysine residue.