Appendix 1—table 2. Fluorescence lifetimes of Alexa647 and Alexa488 coupled to single cysteine variants of hGBP1 determined from ensemble TCSPC.

Fluorescence lifetimes, τ, with corresponding species fractions, x, of Alexa647- and Alexa488-maleimide coupled to different single cysteine variants of the hGBP1 determined by an analysis of the fluorescence intensity decays measured by ensemble TCSPC.

Variant	Alexa647(a)								Alexa488(a)
	Alexa647(a)		Alexa647(a)		Alexa647(a)		Alexa647(a)		Alexa488(a)		Alexa488(a)		Alexa488(a)
	${\displaystyle {\tau }_1}$/ ns	(x1)	${\displaystyle {\tau }_2}$/ ns	(x2)	${\displaystyle {\tau }_3}$/ ns	(x3)	${\displaystyle {⟨\tau ⟩}_x}$/ ns(b)	ΦF,A(c)	${\displaystyle {\tau }_1}$/ ns	(x1)(a)	${\displaystyle {\tau }_2}$/ ns	(x2)	${\displaystyle {⟨\tau ⟩}_x}$/ ns(b)	ΦF,D(c)
N18C	1.85	(0.39)	1.22	(0.49)	0.10	(0.12)	1.33	0.40	4.15	(0.82)	1.35	(0.18)	3.65	0.82
Q254C	2.23	(0.58)	1.42	(0.42)			1.89	0.57	3.60	(0.69)	0.53	(0.31)	2.65	0.59
Q344C	2.06	(0.58)	1.09	(0.42)			1.75	0.52	3.80	(0.94)	1.00	(0.06)	3.63	0.81
T481C	1.89	(0.43)	1.32	(0.57)			1.57	0.43	3.78	(0.93)	1.07	(0.07)	3.59	0.81
A496C	1.21	(0.43)	1.88	(0.57)			1.59	0.48	3.89	(0.84)	1.14	(0.16)	3.44	0.77
Q525C	1.93	(0.65)	1.08	(0.35)			1.63	0.49	3.51	(0.80)	0.66	(0.20)	2.94	0.66
V540C	2.33	(0.65)	1.43	(0.35)			2.02	0.60	4.00	(0.85)	1.86	(0.15)	3.67	0.82
Q577C	2.06	(0.49)	1.42	(0.51)			1.73	0.52	4.15	(0.91)	1.49	(0.09)	3.91	0.88

^(a) The number of fluorescence lifetime components corresponds to the minimum number required to sufficiently describe the experimental data, as judged by a ${\displaystyle {\chi }_r^2}$ criterion (Materials and methods, Equation 10).

^(b) ⟨τ⟩_x is the species weighted average fluorescence lifetime ${\displaystyle {⟨\tau ⟩}_x=\sum _i\left(x_i\cdot {\tau }_i\right)}$ .

^(c) ${\displaystyle {\mathrm{\Phi }}_F}$ is the fluorescence quantum yield of the fluorescent dye species estimated by the species averaged fluorescence lifetime ⟨τ⟩_x using ⟨τ⟩_x and ${\displaystyle {\mathrm{\Phi }}_F}$ of the free dyes as a reference; (Alexa647, ⟨τ⟩_x=1.0 ns, ${\displaystyle {\mathrm{\Phi }}_{F,A}=0.32}$) (Alexa488, ⟨τ⟩_x=4.1 ns, ${\displaystyle {\mathrm{\Phi }}_{F,D}=0.92}$).