Abnormal deposition of aggregated wild-type (WT) human transthyretin (TTR) and 
its pathogenic variants is responsible for cardiomyopathy and neuropathy related 
to TTR amyloidosis. The tryptophan (Trp) fluorescence measurements typically 
used to study structural changes of TTR do not yield site-specific information 
on the two Trp residues per TTR protomer. To obtain such information, tryptophan 
labeled with fluorine at the 5 and 6 positions (5FW and 6FW) was incorporated 
into TTR. Fluorescence of 5FW and 6FW-labeled WT-TTR (WT-5FW and WT-6FW) and a 
single-Trp mutant W41Y showed that the photophysics of incorporated fluoro-Trp 
is consistent with site-specific solvation of the indole ring of W41 and W79. 
19F-NMR showed that solvent accessibility depends on both the location of the 
Trp and the position of the fluorine substituent in the indole ring. 
Unexpectedly, differences were observed in the rates of aggregation, with WT-6FW 
aggregating more rapidly than WT-5FW or WT-TTR. Real-time 19F-NMR urea unfolding 
experiments revealed that WT-5FW is kinetically more stable than WT-6FW, 
consistent with the aggregation assay. In addition, structural perturbations of 
residues distant from either Trp site are more extensive in WT-6FW. Notably, 
residues in the dimer interfaces are perturbed by 6FW at residue 79; pathogenic 
mutations in these regions are associated with reduced tetramer stability and 
amyloidogenesis. The differences in behavior that arise from the replacement of 
a fluorine at the 5-position of a tryptophan with one at the adjacent 6-position 
emphasize the delicate balance of stability in the TTR tetramer.