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. Author manuscript; available in PMC: 2010 Apr 24.
Published in final edited form as: Chem Biol. 2009 Apr 3;16(4):372–381. doi: 10.1016/j.chembiol.2009.02.009

Figure 3. Copper-catalyzed Crosslinking of NRPS Proteins.

Figure 3

NRPS proteins TycA, TycB1, VibB and EntB were crosslinked by first converting the carrier proteins of TycA, TycB1, VibB, and EntB from apo to crypto PCPs with crosslinking probes through the one-pot carrier protein modification conditions: 2.0 μM of carrier protein EntB, VibB, TycA, or TycB1, 0.4 mM of a pantetheine analogue 1a, 1c, 1e, 1f, 2a, or 2d, 3.3 μM PanK, 3.3 μM PPAT, and 3.3 μM DPCK, 10 μM B. subtilis Sfp. Then copper-catalyzed cycloaddition of the crypto PCP azides and alkynes were performed under the following conditions: 25 μl of the one-pot carrier protein modification reactions EntB, VibB, or TycA with a pantetheine azide 1a, 1c, 1e, or 1f, 25 μl of the one-pot carrier protein modification reactions TycB1 with corresponding pantetheine alkyne 2a or 2d, 1 mM CuSO4, 1 mM TCEP, and 0.1 mM TBTA ligand. Negative controls (−) consisted of one-pot reactions without Sfp. (A) SDS-PAGE gel-shift analysis of copper-catalyzed crosslinking assays between crypto TycA azide 1c with crypto TycB1 alkyne 2a. (B) SDS-PAGE gel-shift analysis of copper-catalyzed crosslinking assays between crypto EntB azides 1a, 1e, or 1f and crypto TycB1 alkyne 2d. (C) SDS-PAGE gel-shift analysis of copper-catalyzed crosslinking assays between crypto VibB azides 1a, 1e, or 1f and crypto TycB1 alkyne 2d.