A novel synthetic chemistry approach to linkage-specific ubiquitin conjugation† †Electronic supplementary information (ESI) available: Full experimental details are provided. See DOI: 10.1039/c5ob00130g Click here for additional data file.

Site-specific ubiquitin cysteine mutants enable an elegant method for the linkage-specific conjugation of ubiquitins through dibromomaleimides and dibromopyrdazinediones.

ligation independent subcloning using the primers listed in Table 1 (ThermoScientific). The ubiquitin G76C mutant was also cloned from the WT ubiquitin pET2b(+) plasmid into a pNIC28-Bsa4 vector (see Table 1 for primers).
Ubiquitin mutants UbK27C, UbK48C and UbK63C were generated from the WT pNIC28-Bsa4 vector using the QuikChange site mutagenesis kit (Ailgent Technologies) with the primers shown in Table 1. DNA sequencing confirmed the identity of the constructs and the vectors were heat-shock transformed into E. coli BL21 (DE3) cells for expression.

Protein expression and purification
The following method was used for the expression and purification of all the ubiquitin mutants: Cells were grown at 37 °C in 1 L of Luria-Bertani media supplemented with Kanamycin (50 μgL -1 ). Gene expression was induced, once an OD 600 of 0.6 was reached, by addition of 1 mM isopropyl-β-D-thiogalactopyranoside. The culture was maintained for a further 16 h at 22 °C. Cells were harvested by centrifugation at 4,000 g for 30 min at 4 °C. The cells were resuspended into a lysis buffer (20 mL, 50 mM sodium phosphate buffer, pH 7.4, 250 mM NaCl, 25 mM imidazole, 1 mM TCEP) containing DNAse I (0.2 mg) and two tablets of a cocktail of EDTA-free protease inhibitors (Roche). Cell lysis was achieved through sonication (6 x 30 s burst with 1 min cooling intervals). The resultant lysate was centrifuged at 35,000 g for 30 min at 4 °C and the supernatant loaded onto a pre-equilibrated HisTrap HP 5 mL column (GE Healthcare) pre-charged with Ni 2+ . A linear gradient of imidazole, 25 mM to 1 M was applied to elute ubiquitin. Samples were further purified using a Superdex 75 16/60 size exclusion column (GE Healthcare) equilibrated with 50 mM sodium phosphate buffer, pH 7.4, 250 mM NaCl, 1 mM TCEP.
The protein eluted with one peak of a mass of approximately 12 kDa corresponding to a monomer. Fractions containing the protein were pooled, exchanged into a pH 6 buffer (50 mM sodium phosphate buffer pH 6, 75 mM NaCl), using Amicon Ultra devices (Millipore) and concentrated to 1 mg mL -1 . In cases where the protein was stored 1 mM TCEP was added to the buffer, removed by ultracentrifugation (using VivaSpin sample concentrators (GE Healthcare, 5,000 MWCO)) prior to the modifications. The purity and identity of the protein was confirmed by mass spectrometry, with a yield of approximately 8 mgL -1 of cell culture.
The GFPS147C mutant was expressed and purified as previously described.

Maleimide modified ubiquitin
Modification of all cysteine ubiquitin mutants with dibromomaleimide was achieved using the following method. To a solution of cysteine ubiquitin mutant UbXXC (1 mg mL -1 , 100 μL) in sodium phosphate buffer pH 6 (50 mM sodium phosphate, 75 mM NaCl, pH 6), dibromomaleimide (5 μL, 9 mM solution in DMF) was added. This reaction mixture was incubated on ice for 1 h. Analysis using LC-MS showed complete modification of the cysteine ubiquitin mutant.

Ubiquitin-maleimide-Ubiquitin conjugates
Ubiquitin-maleimide-ubiquitin conjugates were all prepared using the following
The bromomaleimide modified ubiqutin was prepared as previously stated, then excess dibromomaleimide was removed by ultracentrifugation using VivaSpin sample concentrators (GE Healthcare, 5,000 MWCO). GFP mutant S147C (2.6 mg mL -1 , 100 μL) in PBS was buffer exchanged by ultracentrifugation into sodium phosphate buffer pH 8 (50 mM sodium phosphate, 75 mM NaCl, pH 8). Both protein samples were concentrated to half their original volume (to give 50 L) and mixed together in a 1:1 volume ratio, to give a final volume of 100 μL. This reaction mixture was incubated on ice for 1 h. Analysis using LC-MS showed the coupling was complete.
This reaction mixture was incubated on ice for 1 h. Analysis using LC-MS showed complete modification of the cysteine ubiquitin mutant.

Ubiqutin-pyridazinedione-GFP conjugates
Ubiquitin-pyridazinedione-GFP conjugates were prepared using the following method. The bromopyridazinedione modified ubiqutin was prepared as above, then excess dibromopyridazinedione was removed by ultracentrifugation using VivaSpin sample concentrators (GE Healthcare, 5,000 MWCO). GFP mutant S147C (2.6 mg mL -1 , 100 μL) in PBS was buffer exchanged by ultracentrifugation into sodium phosphate buffer pH 8 (50 mM sodium phosphate, 75 mM NaCl, pH 8). Both protein samples were concentrated to half their original volume (to give 50 L) and mixed together in a 1:1 volume ratio, to give a final volume of 100 μL. This reaction mixture was incubated on ice for 16 h. Analysis using LC-MS showed the coupling was complete.