Figure 1: A targeted photocrosslinking strategy to identify the binding partner for the Leu71/Leu73 epitope on ubiquitin.

(a) Schematic of crosslinking strategy using mononucleosomes containing H2BssUb* and the catalytic domain of hDot1L. Biotinylated mononucleosomes are incubated with the catalytic domain of hDot1L, ultraviolet irradiated and purified by streptavidin pull-down. Addition of dithiothreitol to the crosslinked mixture severs the ubiquitin-H2B disulfide bond. Ubiquitin interaction partners (hypothetical crosslinks are indicated by red lines) are subsequently identified by analysis of crosslinked species by immunoblotting or MS. (b) Semi-synthesis of H2BssUb* (1). A synthetic peptide corresponding to residues 64–76 of ubiquitin bearing L-photo-Leu at position 71, an E64C mutation and a C-terminal hydrazide moiety, was assembled using Fmoc-SPPS. The ubiquitin fragment Ub(1–63) was heterologously expressed in E. coli as an intein fusion and converted to an α-thioester by thiolysis in vitro. The two ubiquitin fragments were joined by NCL, forming full-length ubiquitin 2a, which was reacted with bromoacetic acid to afford protein 2b. Subsequent oxidation and ligation to cysteamine yielded ubiquitin analogue, 2. Histone H2B bearing a K120C mutation was heterologously expressed in E. coli, and reacted with DTNP, generating the activated asymmetric disulfide species, 3. In the final step, 2 and 3 were allowed to react at pH=6.9 to form the disulfide–linked H2BssUb*, protein 1. (c) RP-HPLC and ESI-MS analysis of the final protein 1 (MW: 22,458 Da calculated, 22,457.5 Da observed). (d) Ethidium-bromide-stained native gel of reconstituted mononucleosomes containing H2BssUb*.