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. Author manuscript; available in PMC: 2014 Aug 16.
Published in final edited form as: J Am Chem Soc. 2013 Feb 14;135(8):2999–3010. doi: 10.1021/ja306361q

Figure 3. E-to-Q mutants of SecA do not kinetically trap ATP.

Figure 3

Competitive displacement of MANT-labeled nucleotides by unlabeled nucleotides was monitored kinetically using fluorescence anisotropy spectroscopy at 352 nm in KEMT buffer. WT-BsSecA (panel A), WT-EcSecA (panel B), E208Q-BsSecA (panel C), or E210Q-EcSecA (panel D) at 250–750 nM was equilibrated with 60 nM Mg-MANT-ATP in KEMT buffer (pH 7.6) at 5°C for 3–5 minutes before adding a > 100-fold excess of unlabeled Mg-ADP to initiate the exchange reaction.