Gel filtration assay for PII binding to NRII mutants. PII (48 μM) and the indicated NRII protein (12 μM) were mixed on ice in reaction mixtures containing 1 mM MgCl2, 50 μM free 2-ketoglutarate, and 0.5 mM free ATP, as indicated in Materials and Methods. The bulk of the reaction mixture was loaded onto a Sephadex G-100 column equilibrated in buffer containing 1 mM MgCl2, 50 μM 2-ketoglutarate, and 0.5 mM ATP and eluted at 4°C. Fractions were collected and analyzed on SDS-15% polyacrylamide gels stained with Coomassie brilliant blue R-250. Fraction numbers are indicated above the lanes, and IN indicates the column input. (A) NRII (I141V); (B) NRII (S227R); (C) NRII (Y302N); (D) NRII (A129T/I221V); (E) NRII (L16R).