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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 4. Strain-Promoted Copper-Free Azide-Alkyne Cycloaddition of NRPS Proteins.

Figure 4

The carrier proteins of TycA, TycB1, VibB, and EntB were converted 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, 1e, 1f or 3, 3.3 μM CoaA, 3.3 μM CoaD, 3.3 μM CoaE, 10 μM B. subtilis Sfp. Then strain-promoted copper-free cycloaddition of the crypto PCP azides and alkynes were performed under the following conditions: addition of 25 μl of the one-pot carrier protein modification reactions EntB, VibB, or TycA with a pantetheine analogue 1a, 1e, 1f or 3 and 25 μl of the one-pot carrier protein modification reactions TycB1 with corresponding pantetheine analogues 1a or 3. Negative controls (-) consisted of one-pot reactions without Sfp. (A) SDS-PAGE gel-shift analysis of strain-promoted copper-free crosslinking assay between crypto TycA azide 1a with crypto TycB1 alkyne 3. (B) SDS-PAGE gel-shift analysis of strain-promoted copper-free click crosslinking assay between crypto TycA alkyne 3 with crypto TycB1 azide 1a. (C) SDS-PAGE gel-shift analysis of strain-promoted copper-free crosslinking assays between crypto EntB azides 1a, 1e, or 1f and crypto TycB1 alkyne 3. (D) SDS-PAGE gel-shift analysis of strain-promoted copper-free crosslinking assays between crypto VibB azides 1a, 1e, or 1f and crypto TycB1 alkyne 3. (E) SDS-PAGE gel-shift analysis of strain-promoted copper-free crosslinking assay between predenatured crypto TycA azide 1a with crypto TycB1 alkyne 3. (F) SDS-PAGE gel-shift analysis of strain-promoted copper-free crosslinking assay between crypto TycAΔ23 azide 1a with crypto TycB1 alkyne 3.