Protein cyclization.
Shown are SDS-PAGE time course analyses of a Ubiquitin cyclization reaction. The employed Ubiquitin substrate was produced with both an N-terminal (AGAFADPLVVEI) and a C-terminal (ELASKDPGAFDADPLVVEI) Connectase recognition sequence. This allows the formation of linear (L1 -L4, formed by 1 -4 Ubiquitin proteins) polymers, which are observed in the early stages of the time course. The N-terminus of a given polymer can be fused to its C-terminus, resulting in cyclic assemblies (C2 -C6, formed by 2 -6 Ubiquitin proteins), which present the end product of the reaction. A lower substrate concentration (A, 10 μM) results in smaller assemblies, and a higher substrate concentration results in larger assemblies (B, 100 μM). The assignment of the gel bands is consistent with LC-MS data (below the gels). The plots were normalized to the most intense Ubiquitin signal (Ub2); the BcPAP peaks are more intense (>100%), despite its relatively low abundance. The molecular masses of the ubiquitin assemblies are 13 kDa (L1), 24 kDa (L2), 34 kDa (L3), 45 kDa (L4), 21 kDa (C2), 32 kDa (C3), 43 kDa (C4), 53 kDa (C5), and 64 kDa (C6).