Fig. 2.

Cross-linking and DEER data between MalFG(P78C)K₂ and sites in the N-lobe of MalE. (A) MalFG(P78C)K₂ in DDM micelles (2.5 µM) was incubated with MalE(G13C) (5 µM) and homobifunctional thiosulfonate cross-linkers (1 mM) in the presence of different cofactors as indicated. The cross-linked products were subsequently analyzed by SDS/PAGE. The cross-linkers EBS, HBS, and PBS have approximate spacer lengths of 5.2 Å, 10.4 Å, and 24.7 Å (see SI Materials and Methods for more details). (B) DEER analysis on MalFGK₂ transporters solubilized in n-Dodecyl β-D-maltoside carrying the spin label at position 78 in MalG incubated with MalE spin-labeled at the N-lobe (position 80) in different nucleotide states in the presence and absence of maltose, as indicated. (Upper) Normalized DEER Form factors F(t). (Lower) Distance distributions obtained with Tikhonov regularization using the software DeerAnalysis2011 (35). The mutants are denoted by the name of the relative subunit (e.g., G or E) and the corresponding residue number.