Table 1 Spectroscopic properties of Cph1 PGP, Cph1 PG, SyB-Cph2 GP and their variants as shown in Figs. 2, 3 and 4.

Construct	λ_max (ab) (nm)	λ_max (ex) (nm)	λ_max,(em) (nm)	Δλ_max, (em–ab) (nm)	Δλ_max, (em—ex) (nm)	Φ_f	τ_mean (ns)
Cph1 PGP WT	658	643	675	17	32	0.024	0.83
Cph1 PGP Y176H	642	643	672	30	29	0.144	1.90
Cph1 PGP Y263F	651	652	678	27	26	0.082	1.26
Cph1 PGP Y263S	642	642	669	27	27	0.125	1.46
Cph1 PGP Y176H/Y263S	642	642	670	28	28	0.176	2.00
Cph1 PGP Y176H/Y263F	642	642	672	30	30	0.160	1.92
Cph1 PG WT	645	645	671	26	26	0.018	0.69
Cph1 PG Y176H	644	644	672	28	28	0.108	1.74
Cph1 PG Y176H/Y263S	643	643	669	26	26	0.171	2.00
SyB Cph2 GP WT	632	631	660	28	29	0.124	2.07
SyB Cph2 GP D86H	631	632	655	24	23	0.202	2.60

Table S2 provides the detailed results for the fluorescence lifetime fits including an exhaustive error analysis. The fluorescence quantum yield Φ_f is given as fraction.

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