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. Author manuscript; available in PMC: 2017 Nov 16.
Published in final edited form as: J Am Chem Soc. 2016 Nov 4;138(45):14832–14835. doi: 10.1021/jacs.6b08656

Figure 2.

Figure 2

Fluoroacetamide of FAcK reacted with thiol of Cys in proximity created by the Afb-Z protein complex. (a) Structure of the Afb–Z protein complex (PDB ID 1LP1), with 36FAcK in the Afb and 6Cys in the Z protein highlighted. (b) Formation of the Afb/MBP-Z covalent complex after incubation of the Afb36FAcK with the MBP-Z6Cys, indicated by the red arrow in a 10% Tricine-SDS-PAGE gel with Coomassie Brilliant Blue staining (Lane 3). NEM: N-ethylmaleimide. + indicates the protein or the reagent added, − not added. (c) Crosslinking reaction of the Afb36AcK with the MBP-Z6Cys. Afb36AcK/MBP-Z6Cys (molar ratio 10/1) in PBS solution was incubated at 37°C for 24 hrs. (d) Crosslinking reaction of the Afb36FAcK with the MBP-Z6Cys or the MBP-Z11Cys. Afb36FAcK/MBP-Z (molar ratio 10/1) in PBS solution was incubated at 37°C for 24 hrs. (e) Concentration dependency for crosslinking reaction of the Afb36FAcK with the MBP-Z6Cys. Molar ratio of Afb36FAcK/MBP-Z6Cys was changed from 0/1 to 20/1 in PBS solution and incubated at 37°C for 21 hrs. (f) Incubation time dependency for crosslinking reaction of the Afb36FAcK with the MBP-Z6Cys. Afb36FAcK/MBP-Z6Cys (molar ratio 10/1) in PBS solution was incubated at 37°C for the specified amount of time. (g) Crosslinking of the Afb36FAcK with the MBP-Z6Cys (molar ratio 10/1) in 50 mM tris (hydroxymethyl) aminomethane buffered saline (pH 8.8) in the presence of 10 mM TCEP (to prevent thiol oxidation) for 24 hrs at 37°C.