Subnanosecond motions of tryptophan residues in proteins

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Abstract
The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 ns, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B(20,000 daltons) has a single rotational correlation time (.vphi.) of 9.9 ns at 20.degree. C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein in (18,000 daltons) exhibits .vphi. of 0.09 and 1.26 ns, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8.degree. C (.vphi. = 31.4 ns), whereas at 43.degree. C it rotates rapidly (.vphi.I = 0.14 ns) within a cone of semiangle 26.degree., in addition to rotating together with the whole protein (.vphi. = 14 ns). Of particular interest is the large angular range (semiangle, 34.degree.) and fast rate (.vphi.I = 0.51 ns) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.