The impact of substrates on the conformational state of TM1a. smFRET data of BasC variant TM1a-12 labeled with Alexa 546 and Alexa 647 (A and B) and TM1a–TM4 variant labeled with sCy3–sCy5 (C and D) in the presence and absence of amino acids. A, C) Representative histograms of apparent FRET E* distributions for TM1a–TM12 and TM1a–TM4 variants under different buffer conditions. Vertical solid lines represent the mean E* value of the apo state and dashed lines that of the respective amino acid. B) Quantitative analysis of the population distribution between low and high FRET for apo and holo conditions in TM1a–TM12. We statistically compared the relative abundance of both states in independent repeats (black dots) by plotting mean ± SEM of the percentage of high-FRET state. Paired t tests were conducted against apo measurements from the same day. P values are indicated by asterisks (****<0.001). D) Quantitative analysis of apparent FRET E* differences between holo and apo states of BasC TM1a–TM4. For this, mean values of the holo distribution were statistically compared with an apo measurement from the same day (ΔE*, black dots). Mean ΔE* ± SEM are displayed, and paired t tests were performed using the absolute E* values comparing apo and holo measurements of the same day. P values are denoted by asterisks (****<0.001 and **<0.01).