Figure 2.

(a) Emission time trace of a doubly labeled TMR/Cy5 SNase molecule immobilized on glass in buffer A with an oxygen scavenging system (donor emission is dotted, acceptor emission is solid). A median filter was applied to reduce the noise caused by triplet state-induced fluorophore blinking. There are large and gradual fluctuations in I_d and I_a that occur over tens of milliseconds. (b) FRET efficiency time trace calculated from a according to E(t) = [1 + γI_d/I_a]⁻¹. The inset shows the autocorrelation of E(t) together with an exponential fit. τ_E is the characteristic time scale of the E(t) fluctuations displayed in this trace. (c) Histogram of E(t) fluctuation time constants τ_E for 100 doubly labeled SNase molecules. The values range from 10 msec to 1 sec, with the average being 41 msec. (d) Histogram of E(t) fluctuation time constants τ_E for doubly labeled SNase in the presence of active-site inhibitor pTp (50 mM pTp, K_d = 100 nM). These time constants are considerably larger (average = 133 msec) than those measured for free SNase. (e) Scatter plot of E(t) fluctuation amplitudes a_E vs. mean energy transfer efficiencies Ē for free SNase (squares) and pTp-bound SNase (circles). The scatter in Ē may reflect the nonspecific nature of Cy5 labeling. The solid line represents the maximum possible contribution of dipole fluctuations of Cy5 to a_E for free SNase. Only the molecules that display large E(t) fluctuations (>70% of the total) are shown. The others are concentrated around the bottom right corner (not shown) and are likely caused by Cy5 labeling at a site close to Cys²⁸.