Figure 5.

Membrane association of various forms of BjPutA. (A) Wild-type oxidized BjPutA, wild-type BjPutA reduced with 20 mM proline, and NPPG-inactivated BjPutA were incubated with 0.8 mg mL⁻¹E. coli polar liposomes for 1 h in 10 mM HEPES buffer (150 mM NaCl (pH 7.5)). Soluble and lipid-associated fractions were then separated by air-fuge ultracentrifugation and analyzed using SDS-PAGE, as previously described (33). (B) Lipid partitioning assays were performed with BjPutA mutant R51E in the presence and absence of 20 mM proline, as described in (A). (C) The ability of the BjPutA R51E mutant to functionally associate with inverted membrane vesicles was measured by incubating BjPutA wild-type or mutant R51E mutant with 50 mM proline, 4 mM o-AB, and 0.1 mg mL⁻¹ vesicles prepared from JT31 putA⁻E. coli. Reactions were performed in 20 mM 3-(N-morpholino)-propanesulfonic acid (pH 7.5) and o-AB/P5C adduct formation monitored at 443 nm.