FIGURE 5.

Experimental and fitted fluorescence correlation curves of YC3.60 after excitation and detection of the acceptor (λ_ex 496 nm, λ_det 510–556 nm). The curves are normalized at t_c = 1.2 μs. (A) Correlation curves for YC3.60 in presence of Ca²⁺. Three different combinations of polarizations are presented: autocorrelation of the fluorescence polarized parallel to the excitation polarization (pp, curve 1), perpendicular to the excitation (ss, curve 2), and cross correlation of both polarizations (ps and sp, curve 3). Only the polarization dependent part of the correlation curves can be assigned to rotational diffusion. The measurement time was 14 min. (B) pp-correlation curves of YC3.60 in presence of Ca²⁺ (curve 1) are compared to YC3.60 in absence of Ca²⁺ (curve 2). The rotational part of curve 1 (+Ca²⁺) can be reproduced well by a single exponential, whereas curve 2 (−Ca²⁺) shows significant deviations and exhibits a second decay time. Fit parameters are presented in Table 3. (C) Residuals for the global fit with: (YC3.60(+Ca²⁺): 3 exponentials, YC3.60(−Ca²⁺): 4 exp.). (D) For comparison residuals for the global fit with: YC3.60(±Ca²⁺): 3 exp; i.e., a single independent rotational correlation time for each of the two curves (fit not shown).