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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 2. Verification of Peptidyl Carrier Protein Modification.

Figure 2

Conversion of the carrier proteins of TycA, TycAΔ23, and TycB1 from apo- to crypto-PCP with the panel of crosslinking probes 1–3 was accomplished using the one-pot carrier protein modification conditions: 2.0 μM of carrier protein TycA, TycAΔ23, or TycB1, 0.4 mM of a pantetheine analogue 1–3, 3.3 μM PanK, 3.3 μM PPAT, and 3.3 μM DPCK, 10 μM B. subtilis Sfp. Verification of the modification was established by tagging the crypto-PCP with the corresponding coumarin alkyne 4 or coumarin azide 5 using the following conditions for the cycloaddition protocol: addition of 50 μM coumarin alkyne 4 or azide 5, 1 mM CuSO4, 1 mM tris(2-carboxyethyl) phosphine (TCEP), and 0.1 mM tris-(benzyl triazolylmethyl)amine (TBTA) ligand. For the reaction of cyclooctyne 3 with 5, CuSO4, TCEP, and TBTA were omitted from the reaction and all other variables were kept constant. Negative controls (−) consisted of one-pot reactions without Sfp. The fluorescently tagged crypto PCPs were detected by UV visualization on SDS-PAGE gels. (A) SDS-PAGE gel of fluorescently tagged crypto TycA azides 1a-f. (B) SDS-PAGE gel of fluorescently tagged crypto TycA alkynes 2–3. (C) SDS-PAGE gel of fluorescently tagged crypto TycB1 azides 1a-f. (D) SDS-PAGE gel of fluorescently tagged crypto TycB1 alkynes 2–3. (E) SDS-PAGE gel of fluorescently tagged crypto TycAΔ23 azides 1a-f. (F) Structures of fluorescent coumarin alkyne 4 and azide 5.