Figure 2.
Intramolecular FRET reveals a closed conformation for α-synuclein. A, The FRET of 400 nm recombinant YFP-synuclein-CFP (YsynC), CFP with YFP (C+Y), FlAsH-synuclein-CFP (FsynC) and FlAsH-synuclein with synuclein-CFP (Fsyn+synC) fusion proteins in 20 mm NaPO4, pH 7.4, was measured using a 96-well plate reader. Both YsynC and FsynC show significant intramolecular FRET, although to different extents. In contrast, there was no intermolecular FRET detected between C and Y or between Fsyn and synC. The values indicate mean ± SEM, n = 6–16 wells per condition. B, The FRET of α-synuclein fusion proteins was determined without (filled bars) or with 7.5 m urea (open bars) in 20 mm NaPO4, pH 7.4. Urea reduces the intramolecular FRET of FsynC and YsynC but not the direct synuclein-CFP-FlAsH fusion synCF. Data show mean ± SEM; n = 6–8 wells per group. C, Wild-type and mutant FsynC fusion proteins were incubated with increasing concentrations of NaCl (0–400 mm) in 1 mm NaPO4, pH 7.4. In all cases, intramolecular FRET increases at lower salt concentrations, but the increase is greater for FE46KC, and less for FA30PC and FA53TC relative to wild-type FsynC. Note the higher FRET values than in other panels because of the lower NaPO4 concentration (1 vs 20 mm in A and B, and 3 mm in D). The values indicate mean ± SEM; n = 7–12 wells per condition. D, Wild-type and A30P mutant FsynC fusion proteins were incubated without (black) or with (gray) 400 μm membranes containing 10% cholesterol, 33% sphingomyelin, 33% brain phosphatidylserine and 23% brain phosphatidylcholine (binding lipids) in the absence or presence of 400 mm NaCl and 3 mm NaPO4, pH 7.4. In low salt, the A30P mutation inhibits the reduction in FRET but has no substantial effect in the presence of 400 mm NaCl. The values indicate mean ± SEM; n = 7–8 wells per condition.