Correlation of tryptophan fluorescence intensity decay parameters with proton NMR-determined rotamer conformations: [tryptophan2]oxytocin

Abstract

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 [tryptophan2]oxytocin at pH 3 are presented and compared with 1H 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 1H NMR-determined chi 1 rotamer populations of the indole side chain. 15N 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.

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

“…A distribution of decay times centred around three mean exponential values describes the decay kinetics irrespective of the sample state. This behaviour of tryptophan is presently thought to be a consequence of the combined effect of the three discrete rotamers of tryptophan [28][29][30] and solvent interaction in the 1 L a fluorescing state. [31] The sharpening of the three peaks on immobilisation suggests the rotamers are restricted perhaps due to structural rigidity, which is more pronounced after transferring into organic media.…”

Dynamics of Arrestin-Rhodopsin Interactions

“…, or the intensity-weighted average fluorescence lifetime (28,60,61). These values represent the mean of the two or three reported sets of lifetimes Ϯ S.E.…”

Characterization of Single-Tryptophan Mutants of Histidine-Containing Phosphocarrier Protein: Evidence for Local Rearrangements during Folding from High Concentrations of Denaturant

“…As a consequence of the existence of different rotameric states of the indole moiety, a tryptophan residue can possess multiple fluorescence lifetimes. Each state is represented by a discrete excited-state lifetime (32), and in a protein the distribution over these states and the value of the lifetime depend on the protein structure and the microenvironment in which the tryptophan residue is located. Analysis of the fluorescence intensity decay results in a distribution of fluorescence lifetimes; the analysis of the fluorescence anisotropy decay produces a distribution of correlation times associated with different tryptophan rotational modes (19).…”