Fluorescence properties of recombinant tropomyosin containing tryptophan, 5-hydroxytryptophan and 7-azatryptophan

Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptopha n (5OH122W) or 7-azatryptophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, correspo nding to a solvent-exposed position c of the coiled-coil heptapeptide repea t. The emission spectrum of the probe in each mutant is blue-shifted slight ly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2-3 ns while the flu orescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexpone ntial fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential, The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptop han (when emission is collected from the entire band) are single exponentia l. Incorporation into tropomyosin induces triple-exponential fluorescence d ecay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7- azatryptophan. The range of lifetimes observed for 5-hydroxyindole and 5-hy droxytryptophan at high pH and in the nonaqueous solvents were used as a ba se with which to interpret the lifetimes observed for the 5OH122W and indic ate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 5OH122W.