Table1: Fluorescence parameters for IDP/polyproline samples.

Photophysical parameters: δ is the correction factor for acceptor direct excitation; τ_D is the donor-only fluorescence lifetime (from tail fits); α is the correction factor for spectral cross-talk; τ_A it the acceptor fluorescence lifetime (from tail fits); r_ss(donor) is the donor steady-state anisotropy of the donor-only subpopulation; and r_ss(acceptor) is the stead-state anisotropy of the acceptor-only subpopulation upon acceptor direct excitation. Note that δ increases upon coupling to proteins, an indication that the use of free dyes would result in non-representative correction factors. After the correction factors are applied, the mean fluorescence stoichiometry ratio of the reference data set is 〈S〉 = 0.501 ± 0.004. The decreased donor lifetimes of the HIV–1 Integrase sample result in a substantially decreased fluorescence stoichiometry ratio. Replacing the tryptophan near the donor fluorophore (IN-WDA) with a phenylalanine (IN-FDA) leads to an increase in donor lifetime and fluorescence stoichiometry ratio. A similar effect is apparent when the labeling positions of the donor and acceptor are swapped (IN-WAD). Color code as in Figure 4.

Sample		δ	τD	α	τA	rss(donor)	rss(acceptor)	〈E〉	〈S〉
Free-Dyes		0.035	3.92	0.041	3.89	*	*	-	-
Reference Data Set	P40	0.045	3.998	0.053	3.939	0.019	0.016	0.175	0.504
	P27	0.040	3.994	0.043	3.851	0.006	0.015	0.336	0.503
	P20	0.040	3.948	0.048	3.937	0.011	0.011	0.563	0.502
	P14	0.050	3.794	0.020	3.899	0.022	0.007	0.774	0.502
	P11	0.038	3.983	0.047	3.898	0.021	0.020	0.863	0.497
	P6	0.045	3.951	0.020	3.767	0.020	0.016	0.952	0.506
	sNh−	0.043	3.984	0.053	4.288	0.045	0.026	0.529	0.501
	sNh+	0.040	3.966	0.051	4.001	0.038	0.014	0.318	0.494
average		0.043	3.95	0.042	3.94	0.023	0.016	-	0.501
standard deviation		0.004	0.07	0.014	0.15	0.013	0.006	-	0.004
Integrase Data Set	IN-WDA	0.042	3.37	0.055	4.11	0.049	0.021	0.654	0.435
	IN-FDA	0.057	3.66	0.047	4.09	0.030	0.031	0.649	0.480
	IN-WAD	0.044	3.81	0.047	4.26	0.032	0.038	0.633	0.506

^*defined as zero for the purpose of correcting for differential collection efficiencies of the parallel and perpendicular detection channels

^-orange values in the Integrase Data Set are more than 3 standard deviations away from the mean of the Reference Data Set.