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. 2011 Feb 21;47(7):2026-8.
doi: 10.1039/c0cc04868b. Epub 2011 Jan 6.

Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups

Carlos A Castañeda  1 , Jia Liu, Tanuja R Kashyap, Rajesh K Singh, David Fushman, T Ashton Cropp
Affiliations

Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups

Carlos A Castañeda et al. Chem Commun (Camb). .

Abstract

E2 enzymes catalyze the ATP-dependent polymerization of polyubiquitin chains which function as molecular signals in the regulation of numerous cellular processes. Here we present a method that uses genetically encoded unnatural amino acids to halt and re-start ubiquitin polymerization providing access to natural-linkage, precision-length ubiquitin chains that can be used for biochemical, structural, and dynamics studies.

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Figures

Fig. 1
Fig. 1
SDS-PAGE gels showing the corresponding polymerization reactions with and without E1 as controls. (A) Full-length monomeric Ub substrates: K48Lys(Boc), K48R, K48Lys(Boc) treated with TFA, and wild-type. (B) Combinations of UbK48Lys(Boc) and Ub1–74 halt polymerization at di-Ub (Ub2) and can be iterated to produce tri-Ub (Ub3).
Fig. 2
Fig. 2
Overlay of {15N–1H} TROSY-HSQC spectra of TFA-treated Ub1–74 K48Lys(Boc) monomer (red) and Ub1–74 monomer (blue).
Scheme 1
Scheme 1
Method to generate poly-Ub with natural linkages and defined length. Blue circle represents Ub with a C-terminal terminator (e.g. UbD77 or Ub1–74). Orange circle represents UbK48Lys(Boc) variant. The trimer can be then be treated with TFA and extended to longer lengths with more rounds of enzymatic assembly, as shown above.
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