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. Author manuscript; available in PMC: 2016 Dec 4.
Published in final edited form as: J Mol Biol. 2015 Oct 30;427(24):3890–3907. doi: 10.1016/j.jmb.2015.10.015

Figure 3. Disulfide cross-linking to probe interactions between P1 and P4.

Figure 3

(A) Disulfide cross-linking between P1*(H45C) to P4*(S492C) for P1* and either P3P4* or P3P4P5* (stars denote Cys-modified proteins). Left: Quantification of P1*-P4* cross-linked band on SDS-PAGE for P1* (12 µM) reacting with P3P4* or P3P4P5* (24 µM) at the indicated temperatures for 1 hour. Addition of 2mM ADP or ATP reduces P1*-P4* cross-linking, with ATP having the greater effect. Right: SDS-PAGE of P1* cross-linked to either P3P4P5* or P3P4* after 1 hour at 55 °C. P1* and P4* give no cross-linking under identical conditions. (B) Disulfide cross-linking for CheAFL* (AFL*) and 41AA containing the H45C and S492C Cys substitutions Left: Comparison of trans P1*-P4* cross-linking within the CheAFL (P1*P4*) homodimer to cis cross-linking within the 41AA* (P1*P4*) homodimer. ATP reduces cross-linking with 41AA, but not with AFL. Right: SDS-PAGE of CheAFL* and 41AA* (12 µM) cross-linked dimers after 1 hour at 55 °C: P1* mutant homodimers form only P1*-P1* bands, P4* mutants form only P4*-P4* bands, whereas P1*P4* double mutants form all three cross-link types: P1*-P1*, P1*-P4* and P4*-P4*. P4*-P4* crosslinks appear reduced in the double mutants, which favor P1*-P4* cross-linking. (C) Exchange of P1* and P4* homodimers at 55 °C overnight (Ex) rescues P1*-P4* cross-linking in CheAFL* but not in 41AA*. Left: SDS-PAGE of CheAFL* (6 µM) + CheW (6 µM) cross-linking at 55 °C for 1 hour; the exchanged (Ex) P1* and P4* homodimers produce the same banding pattern as the double mutant (P1*P4* = H45C,S492C) for CheAFL*. Right: SDS-PAGE of P1* and P4* exchange for 41AA* (6 µM), which fails to form the P1*-P4* cross-link upon mixing of the single mutants (third lane) or after many hours of exchange at 55 °C (last lane). Lane 1 and Lane 2 show that overnight subunit exchange at 55 °C does not affect cross-linking of the double mutant. Cross-linking was performed by adding 5 mM Cu(II)phen3 at 55 °C for 1 hr whether or not preceded by an exchange period. (D) Quantified band intensities of the cross-linked CheA* (6 µM) with and without CheW (6 µM) and Tm14 (18 µM) at 55 °C for 1 hour. Tm14 and CheW inhibit P1*-P4* cross-linking in all cases.