Picosecond dynamics of a peptide from the acetylcholine receptor interacting with a neurotoxin probed by tailored tryptophan fluorescence.

A tryptophan analog, dehydro-N-acetyl-L-tryptophanamide (Delta-NATA), which is produced enzymatically via L-tryptophan 2',3'-oxidase from Chromobacterium violaceum, is newly used for time-resolved fluorescence. The absorption and emission maxima of Delta-NATA at 332 and 417 nm, respectively, in 20% dimethylformamide-water are significantly shifted to the red with respect to those of tryptophan in water, permitting us to measure its fluorescence in the presence of tryptophan residues. We demonstrate that the steady-state spectra and the fluorescence decay of Delta-NATA are very sensitive to environment, changing dramatically with solvent as the chromophore is localized within a protein and when this tagged protein binds to a peptide. The tryptophan oxidase was also used to modify the single Trp of a neurotoxin from snake (Naja nigricollis) venom. Modification of the toxin alpha (dehydro-tryptophan-toxin alpha) permitted its investigation in complex with a synthetic 15-amino acid peptide corresponding to a loop of the agonist-binding site of acetylcholine receptor (AchR) from Torpedo marmorata species. The peptide alpha-185 possesses a single Trp at the third position (Trp187 of AchR) and a disulfide bridge between Cys192 and Cys193. A single-exponential rotational diffusion time with a constant of 1.65 ns is measured for the isolated 15-amino acid peptide. This suggests that Trp motion in the peptide in solution is strongly correlated with the residues downstream the peptide sequence, which may in part be attributed to long-range order imposed by the disulfide bond. The dynamics of the bound peptide are very different: the presence of two correlation times indicates that the Trp][87 of the peptide has a fast motion (tau(r1) = 140 ps and r(0)(1) = 0.14) relative to the overall rotation of the complex (tau(r2) = 3.4 ns and r(0)(2) = 0.04). The correlation of the Trp residue with its neighboring amino acid residues and with the overall motion of the peptide is lost, giving rise to its rapid restricted motion. Thus, the internal dynamics of interacting peptides change on binding.