Larger conformational change in UN2A occurs at pH 6.0 with S100A1 binding. FRET experiments were performed at pH 6.0 using the same conditions as the initial experiments at pH 7.4. (A) Similar to pH 7.4, there was no significant differences were observed in low Ca²⁺ conditions. (B) Under high Ca²⁺ conditions, there was an increase in the CFP (465 nm) peak and a decrease in the YFP (527 nm) peak, though the magnitude of the change is greater than was observed at pH 7.4, suggesting that there is a greater conformational shift with S100A1 binding at pH 6.0. (C) Fluorescence spectra of UN2A-FRET clone in presence of S100A1 at pCa 3 at pH 7.4 and pH 6.0 overlaid to highlight the shift in the FRET signal at lower pH. There is a larger CFP (465 nm) peak and smaller YFP (527 nm) peak at pH 6.0, consistent with decreased FRET.