Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy

The dynamics of the binding reaction of ANS to native and partly folded (mo lten globule) tuna and horse apomyoglobins has been investigated by fluores cence correlation spectroscopy and frequency domain fluorometry. The reacti on rate has been measured as a function of apomyoglobin and ANS concentrati ons, pH, and temperature. Examination of the autocorrelation functions show s that the reaction rate is fast enough to be observed in tuna apomyoglobin , whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyogl obin at pH 7 and room temperature the on rate is 2200 muM (-1) s (-1) and t he off rate is 5900 s (-1), in comparison with k(on) = 640 muM (-1) s (-1) and k(off) = 560 s (-1) for horse myoglobin as measured previously. The ind ependence of the reaction rate from the ANS concentration indicates that th e reaction rate is dominated by the off rate. The temperature dependence of the on-rate shows that this rate is diffusion limited. The temperature dep endence of the off rates analyzed by Arrhenius and Ferry models indicates t hat the off rate depends on the dynamics of the protein. The differences be tween horse and tuna apomyoglobins in the ANS binding rate can be explained in terms of the three-dimensional apoprotein structures obtained by energy minimization after heme removal starting from crystallographic coordinates . The comparison of the calculated apomyoglobin surfaces shows a 15% smalle r cavity for tuna apomyoglobin. Furthermore, a negative charge (D44) is pre sent in the heme cavity of tuna apomyoglobin that could decrease the streng th of ANS binding. At pH 5 the fluorescence lifetime distribution of ANS-ap omyoglobin is bimodal, suggesting the presence of an additional binding sit e in the protein. The binding rates determined by FCS under these condition s show that the protein is either in the open configuration or is more flex ible, making it much easier to bind. At pH 3, the protein is in a partially denatured state with multiple potential binding sites for ANS molecule, an d the interpretation of the autocorrelation function is not possible by sim ple models. This conclusion is consistent with the broad distribution of AN S fluorescence lifetimes observed in frequency domain measurements.