There has been a great interest in understanding ubiquitin (Ub) and its cellular signaling recognition of ubiquitin binding domains (UBDs). Ubiquitin binding domains have moderate to low affinity binding for monomeric Ub (monoUb) and polymeric Ub (polyUb) chains, hindering the understanding of Ub and its interaction with UBDs. Here we report the incorporation of a genetically encoded photocrosslinker, p-Benzoyl-L-phenylalanine (Bpa) into Ub, via an orthogonal suppressor tyrosyl tRNA (tRNATyr) and Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyRS) pair. Fourteen different residues of Ub were mutated to an amber stop codon, TAG, that base pairs with the anticondon of the tRNATyr, thus placing Bpa at its desired position in Ub. Incorporation of Bpa into Ub helps enhance the functionality of Ub by allowing Ub to form a convalent bond with UBDs under UV exposure. We report the crosslinking (covalent bond formation) of monoUb and K48 and K63 enzymatically linked diUb chains to, UBDs, human ubiquitin-associate 2 domain (UBA2) and Rap 80. This approach gives us the ground work for understanding Ub’s cellular signaling recognition of UBDs.

Photoinduced interaction of ubiquitin binding domains with genetically encoded p-Benzoyl-L-phenylalanine monomeric ubiquitin and polyubiquitin chains

254th National Meeting (2017)

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