Fluorotryptophan Incorporation Modulates the Structure and Stability of Transthyretin in a Site-Specific Manner

Abstract

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 emphasizes the delicate balance of stability in the TTR tetramer.

Kinetic analysis of the multistep aggregation pathway of human transthyretin

“…S1. The translational diffusion coefficient of C10S-S85C was measured on the 900-MHz spectrometer using 15 N-filtered DOSY at pH 7.0 and 298 K. The measured translational diffusion coefficient is (5.1 ± 0.1) × 10 −7 cm 2 /s for C10S-S85C, which agrees with the reported data of WT TTR as (5.1 ± 0.2) × 10 −7 cm 2 /s (46). BTFA labeling is site-specific to S85C because the observed tetramer 19 F peak at −84.19 ppm in TTR F at 298 K is absent in a C10S control that was treated with BTFA the same way as C10S-S85C (SI Appendix, Fig.…”

“…Protein Expression, Purification, and Labeling. TTR mutants were expressed as described previously (46). See SI Appendix for details of protein purification and labeling.…”

Prognostic impact of serum transthyretin in patients with non‑small cell lung cancer

“…Thus, the results of the current study revealed, for the first time, that preoperative TTR was an independent prognostic factor for 5-year recurrence-free survival in NSCLC patients. The normal function of TTR is to transport thyroxine and RBP/retinol complex in the blood (14). TTR exists in the blood as a homotetramer and contains four tryptophans which are essential amino acids (5).…”

Mixed Fluorotryptophan Substitutions at the Same Residue Expand the Versatility of 19F Protein NMR Spectroscopy

“…Such mixtures may be readily achieved by mixing of different isoforms or through providing multiple fluoroindole precursors during biosynthesis. The value of applying F‐Trp mixtures also follows from dramatically different 19 F chemical shift dispersion for 5F‐ versus 6F‐Trp for the two native Trp residues of transthyretin, exhibiting distinct sensitivity to environment despite unperturbed folding, as well as a greater 4F‐Trp chemical shift dispersion versus 5F‐ or 6F‐Trp in lysozyme . Multi‐site perturbation should, therefore, be straightforwardly and efficiently tracked by 1D 19 F NMR experiments following multiple F‐Trp types instead of relying upon a single F‐Trp type which may or may not be an effective probe in all conditions.…”

“…In summary, mixtures of membrane protein isoforms that contain differently‐substituted F‐Trp residues at each Trp position were readily biosynthesized. Although the focus here has been on 19 F NMR spectroscopy, it should also be noted that the modified photophysics of F‐Trp versus Trp residues imply broader applicability of these approaches for fluorescence spectroscopy and resonance energy transfer characterization. Both 19 F chemical shift and spin dynamics were consistent between AR constructs as a function of position.…”