Figure 5.

The conformational change of 44OOC-5 is dependent on ADP only under reducing condition. Limited proteolysis of 44OOC-5 wild type by trypsin in the presence of nucleotides and under different redox conditions. Protein was first incubated in 20 mM Tris-HCl, 200 mM NaCl, pH 8.5, 4% glycerol with nucleotide (2.5 mM with 5 mM MgCl₂) in oxidized (A) or reduced buffer with 2 mM TCEP (B) for 30 min at 20°C and then digested by trypsin at 20°C for 10 and 30 min. The digested samples were resolved by a reducing 10% Tris-Tricine SDS-PAGE and Coomassie blue stain. (C) Nucleotides ADP and ATP were titrated in reduced buffer with 2 mM TCEP for wild-type 44OOC-5 before trypsin digestion.