AbstractWe have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin (M-TTR), a protein involved in the transport of the thyroid hormone and retinol and associated with various forms of amyloidosis, extending our analysis to the second order derivative of the spectra. This procedure allowed to identify three peaks readily assigned to Trp41, as the three peaks were also visible in a mutant lacking the other tryptophan (Trp79) and had similar FRET efficiency values with an acceptor molecule positioned at position 10. The wavelength values of the three peaks and their susceptibility to acrylamide quenching revealed that the three corresponding conformers experience different solvent-exposure, polarity of the environment and flexibility. We could monitor the three peaks individually in urea-unfolding and pH-unfolding curves. This revealed changes in the distribution of the corresponding conformers, indicating conformational changes and alterations of the dynamics of the microenvironment that surrounds the associated tryptophan residue in such transitions, but also native-like conformers of such residues in unfolded states. We also found that the amyloidogenic state adopted by M-TTR at mildly low pH has a structural and dynamical microenvironment surrounding Trp41 indistinguishable from that of the fully folded and soluble state at neutral pH.