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Perfusion-induced FTIR difference spectra of LacY G46W/G262W in the absence of NPG obtained from samples equilibrated at pH 7 subtracted from the samples equilibrated at pH 8.0 (A,a), 10.5 (A, b), 10.9 (A, c), and 11.5 (A, d), respectively. Difference spectra of the LacY G46W/G262W/E325A variant for the step from to pH 7.0 to pH 11.5 (B, e) and 10.9 (B, f), and for the simple LacY E325A mutant for the step from pH 6.0 to pH 10.0 (C, g).
