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ABSTRACT
The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain [chi]^sub 1^ rotamer populations of the peptides were determined by one-dimensional and two-dimensional ^sup 1^H-NMR. All peptides have a single backbone conformation. The [straight phi]-, [psi]-angles and [chi]^sub 1^ rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5°C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined [chi]^sub 1^ rotamer. In five peptides the [chi]^sub 1^ = -60° rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the [chi]^sub 1^ = 180° rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.
INTRODUCTION
The environmental sensitivity of tryptophan fluorescence is reasonably well understood in simple model systems (Callis, 1997; Chen and Barkley, 1998). Ideally, this knowledge could be used to interpret or predict intrinsic protein fluorescence in terms of protein structure around the tryptophan residues. Individual tryptophans in polypeptides often have multiexponential fluorescence decays. Although excited-state reactions are still invoked (Hudson et al., 1999; Ladokhin, 1999; Moncrieffe et al., 2000; Nanda and Brand, 2000), the multiple fluorescence lifetimes are generally attributed to ground-state heterogeneity (Donzel et al., 1974; Dahms et al., 1995; Hellings et al., 2003). The emitting state of the indole chromophore has several nonradiative decay channels, including intersystem crossing (Volkert et al., 1977; Robbins et al., 1980), solvent quenching (McMahon et al., 1992), excited-state proton transfer (Vander Donckt, 1969; Saito et al., 1984; Yu et al., 1992), and excited-state electron transfer (Petrich et al., 1983; Shizuka et al., 1988; Chen et al., 1996; Chen and Barkley, 1998). Neither the ground-state conformations causing the multiple lifetimes nor the quenching mechanisms governing those lifetimes are known for proteins. Recent efforts have...