Figure 4.

Brightness and correlation amplitudes of the heterotrimer DA₂. (A) The red-channel brightness of the heterotrimer DA₂ is shown as a function of the average FRET efficiency for two limiting cases, one where both mCherries are equivalent (solid line), and one where all energy is transferred to a single mCherry (dotted line). The deviation between the two extreme cases is smaller than the typical experimental uncertainty. The red-channel brightness of the fusion protein Ch-G-Ch (diamonds) agrees, within experimental error, with either limiting case. (B) The red-channel (diamonds), green-channel (exes), and cross-channel (triangles) correlation curves of Ch-G-Ch are analyzed as described in the legend for Fig. 3B. Note that the correlation amplitude of the red channel is lower than that of the green channel, as theoretically predicted by the two-state model. The cross-correlation amplitude is also correctly predicted by the model as summarized in Table 2.