For in vitro spectroscopy of recombinant Twitch-2B, the protein was purified from E. coli using Ni-NTA resin, as described (6). Spectroscopy was performed on a Cary Eclipse Spectrophotometer (Varian). Donor dequenching of Twitch-2B was performed by digesting Twitch-2B in a calcium-bound state overnight at room temperature with chymotrypsin (70 U/ml; Sigma-Aldrich) while recording FRET. A small remaining cpVenuscd emission after chymotrypsin digestion was selectively photobleached (5 min) with an array of six Luxeon Lumiled light-emitting diodes peaking at 530 nm, for a total power dissipation of 14.7 W and 870 lumen. A 500-nm LP (long-pass) filter was used to protect mCerulean3 from bleaching. Calcium-bound Twitch-6 was very resistant to protease digestion. Therefore, EGTA (final concentration, 5 mM) was added during chymotrypsin digestion to obtain the spectrum of dequenched mCerulean3.

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