CORRELATION OF TRYPTOPHAN FLUORESCENCE INTENSITY DECAY PARAMETERS WITH H-1 NMR-DETERMINED ROTAMER CONFORMATIONS - [TRYPTOPHAN2]OXYTOCIN

While the fluorescence decay kinetics of tyrosine model compounds [Laws, W. R., Ross, J. B. A., Wyssbrod, H. R., Beechem, J. M., Brand, L., & Sutherland, J. C. (1986) Biochemistry 25, 599-607] and the tyrosine residue in oxytocin [Ross, J. B. A., Laws, W. R., Buku, A., Sutherland, J. C., & Wyssbrod, H. R. (1986) Biochemistry 25, 607-612] can be explained in terms of heterogeneity derived from the three ground-state chi-1 rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from chi-2 rotations. In this paper, the time-resolved and steady-state fluorescence properties of [tryptophan 2]oxytocin at pH 3 are presented and compared with H-1 NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength and a global analysis of these decay curves for common emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the H-1 NMR-determined chi-1 rotamer populations of the indole side chain. N-15 was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the chi-1 rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two chi-2 populations. Depending upon the degree of correlation between chi-2 and chi-1, there may be from three to six side-chain conformations for the tryptophan residue. The combined fluorescence and NMR results are consistent with a rotamer model in which either (i) the chi-2 rotations are fast compared to the fluorescence intensity decay of the tryptophan residue, (ii) environmental factors affecting fluorescence intensity decay properties are dominated by chi-1 interactions, or (iii) the chi-2 and chi-1 rotations are highly correlated.