Table 1.

Dns-labeled Variants, Foldons, and Characteristics of their Native, CL-bound and Unfolded States from Integrated Fluorescence and TR-FRET measurements^a

Variant	Substructure Probed	Foldonb	Unit’s Unfolding ΔG (kcal/mol)b	CαD - FeA Distance (Å)c	Dns-Heme Distances (Å)d			λmax (nm)h
					Ne	CLf	UGuHClg	Ne	CLf
Dns28	Loop A	Green	10	8.4	n.d.i	n.d.i	n.d.i	490	500
Dns39	Loop B	Nested Yellow	6	14.8	21	23	29	493	493
Dns50	50’s helix / loop C	Nested Yellow	6	15.1	23	33	41	493	484
Dns66	60’s helix	Green	10	13.8	20	30	51	488	489
Dns92	C-helix	Blue	12.8	16.3	24	40	52	494	480
Dns99	C-helix	Blue	12.8	15.2	23	29	50	479	490
Dns104	C-helix	Blue	12.8	18.9	23	35	46	492	490

^aMeasured at 2 hours after mixing.

^bFrom NMR NHX experiments.¹⁵

^cCalculated from the crystal structure, 1HRC.⁵⁸ The Dns fluorophore adds about 6 to 8 Å to this distance.⁵⁹

^dGiven by the first moment of the experimental P(r) distributions from TR-FRET measurements. At distances longer than 1.5×R₀=59 Å, energy transfer rate constants and D-A distances cannot be determined reliably; the structures with r ≥ 1.5 R₀ are treated as having r=1.5×R₀ and thus experimental distributions P(r) for more distant labeling sites yield apparent distances shorter than separation between Dns and the heme in the actual polypeptides.^{35, 59}

^eIn a 25 mM HEPES buffer at pH 7.4.

^fWith TOCL/DOPC liposomes (50 mol% CL, 750 μM total lipid) in a 25 mM HEPES buffer at pH 7.4 at 3 μM cyt c.

^gIn 6 M GuHCl at pH 7.4.

^hDns emission maxima λ_max for all the variants in the GuHCl-denatured state were 500±2 nm.

ⁱHigh degree of Dns fluorescence quenching in Dns28 did not permit detection of TR-FRET decays with our current instrumentation.