Fig. 5.

E. coli SecA can adopt an open conformation in solution. (a) SecA containing single cysteines at positions 824 or 234, or a mutant lacking cysteines (no cys), were labeled with maleimide fluorescein in the presence of the indicated additions. When nucleotide (0.25 mM) was added, 0.5 mM MgCl₂ was also included. The samples were separated by SDS/PAGE and visualized under UV light. In the lane labeled quench+SDS, the quenching reagent, either DTT or glycine, was added before labeling or crosslinking. (b) In parallel, samples were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, separated by SDS/PAGE, and stained with Coomassie blue. The position of dimer crosslinks is indicated. (c) Modification reactions with SecA containing a cysteine at position 824 were performed as in a in the presence of 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (MLPG, 0.1 mM); 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC, 4.2 mM); dodecyl maltoside (DDM, 0.6 mM); decyl maltoside (DM, 3.6 mM); octyl maltoside (OM, 39 mM); octyl glucoside (OG, 36.4 mM); CYMAL4 (15.2 mM); CYMAL6 (1.12 mM); lauryldimethylamine-N-oxide (LDAO, 2 mM); digitonin (1%); or SDS (0.5%). (d) In parallel, samples were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.