We found that the A227D mutation did not alter the excitation Capmatinib or emission spectrum or pH sensitivity of super ecliptic pHluorin (Figure S2). The A227D mutant retained a sigmoidal fluorescence-voltage relationship with a large increase in amplitude and a small leftward shift in V1/2 (Figure 1C). The kinetics of the fluorescence responses were also similar. We determined that the “on” kinetics of ArcLight in response to a +100mV step were best fit by a double exponential curve (Figure 1D and Figure S3A), with the time

constant (tau, τ) of the fast component of ∼10 ms and τ of the slow component of ∼50 ms (Figure S3B). Super ecliptic pHluorin differs from eGFP at nine positions:

80, 147, 149, 163, 175, 202, 204, 206, and 231, out of 238 residues (Figure S1). All but one (163) of the nine residues have outward-facing side chains on the surface of the FP and many of the nine residues reside on the same side of the beta barrel (Figure 2A) as A227. We wanted to determine which of these nine amino acids are important for the A227D mutation to exert the increase signal in ArcLight. We introduced single point mutations in six variant constructs of ArcLight, replacing the most dissimilar residues in super ecliptic pHluorin MK-2206 clinical trial with those in eGFP: R80Q, D147S, Q149N, F202S, T204Q, and the T206A (Figure 2A). While the R80Q or Q149N mutations did not alter the response magnitude of ArcLight, the D147S mutation caused a large decrease in the signal size, and the F202S and T204Q mutations nearly eliminated the ArcLight response (Figure 2A). Conversely, the T206A mutation increased the signal size. We also explored whether replacing the A227 in ecliptic pHluorin with amino acids other than aspartic acid would modulate the response of the

probe. We found that replacement of A227 with glutamic acid enhanced the ΔF/F response over A227; however, this was a significantly smaller increase than that seen with aspartic acid (Figure 2B). Basic residues (i.e., arginine and lysine) at position 227 eliminated the voltage-dependent fluorescence change (Figure 2B). These findings indicate that the presence of a negative charge at position 227 is essential for the increase in fluorescence response magnitude but the single additional carbon present in the glutamic acid side chain reduces this effect. Replacement of A227 with polar residues (i.e., histidine, glutamine, or asparagine) also improved the fluorescence response, but not as much as aspartic acid (Figure 2B). Moving an FP to different positions along the linker between the CiVS and the phosphatase has been shown to alter the signal size and response speed of the resulting probes (Baker et al., 2008).