1. Volume 16, Issue 4, Pages 372-381 (April 2009)
Crosslinking Studies of Protein-Protein Interactions in Nonribosomal Peptide Biosynthesis 
Gene H. Hur, Jordan L. Meier, Jeremy Baskin, Julian A. Codelli, Carolyn R. Bertozzi, Mohamed A. Marahiel, Michael D. Burkart 
Chemistry & Biology 
Volume 16, Issue 4, Pages (April 2009)
DOI: /j.chembiol
Copyright © 2009 Elsevier Ltd Terms and Conditions

2. Figure 1
Overall Scheme of Crosslinking Assay and Panel of Bioorthogonal Pantetheine Crosslinking Probes
(A) Structural diagram of the crosslinking assay.
(B) Structure of azido pantetheine crosslinking probes.
(C) Structure of alkynyl pantetheine crosslinking probes.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2009 Elsevier Ltd Terms and Conditions

3. Figure 2 Verification of PCP Modification
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 analog 1–3, 3.3 μM PanK, 3.3 μM PPAT, 3.3 μM DPCK, and 10 μM Bacillus 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 TCEP, and 0.1 mM 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.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2009 Elsevier Ltd Terms and Conditions

4. Figure 3 Copper-catalyzed Crosslinking of NRPS Proteins
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 analog 1a, 1c, 1e, 1f, 2a, or 2d, 3.3 μM PanK, 3.3 μM PPAT, 3.3 μM DPCK, and 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.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2009 Elsevier Ltd Terms and Conditions

5. Figure 4
Strain-Promoted Copper-Free Azide-Alkyne Cycloaddition of NRPS Proteins
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 analog 1a, 1e, 1f, or 3, 3.3 μM PanK, 3.3 μM PPAT, 3.3 μM DPCK, and 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 analog 1a, 1e, 1f, or 3 and 25 μl of the one-pot carrier protein modification reactions TycB1 with corresponding pantetheine analogs 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.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2009 Elsevier Ltd Terms and Conditions