Probing the kinetics of tryptophan self-assembly with surface-enhanced Raman scattering and other biophysical techniques

• Ano de defesa: 2025
• Autor(a) principal: Cardozo, Gabriel Conishi
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: amyloid fibrils; automontagem; fibras amiloides; Raman; self-assembling; SERS; triptofano; tryptophan
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/43/43134/tde-23092025-105420/

The formation of amyloid fibrils by proteins has been extensively studied and more recently, amyloid-like fibril structures have been observed for single metabolites, including amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine(Tyr). These newly identified ordered aggregates exhibit several hallmarks of protein-derived amyloid fibrils, including electron diffraction patterns, binding to fluorescence probes, cytotoxicity, and a possible link to disease. Despite advances in understanding the formation of these self-assembled structures, there remains a lack of experimental techniques capable of directly detecting the involved intermolecular interactions in a time-dependent manner. In this work, we show that a low-cost and label-free methodology based on surface-enhanced Raman scattering (SERS) enables direct probing of these interactions during the self-assembly process. Using this approach, we first investigated the kinetics of Trp self-assembly in pure water. We detected directly, at the molecular level, the formation of hydrogen bonds, -stacking, and hydrophobic interactions. Hydrogen bonds were evidenced since the first day of self-assembly by changes in the relative Raman intensities of bands at 1638, 1422, and 1039 cm1, with the main vibrational contributions attributed to the carboxylate and amino groups. These changes indicate the formation of initial dimers, tetramers, or even higher-order supramolecular structures. The bands at 1638 and at 1039 cm1 were absent in the monomeric Trp spectrum and could therefore be considered as SERS markers of amyloid-like fibril. After the first day of self-assembly, several spectral changes correlated to perturbations of the -electron system were also observed. Among these are the splitting of the band at 492 cm1 and the downshifts of the bands at 860 and 1230 cm1. These spectral changes were reproduced in a calculated dimer forming a - interaction. Furthermore, these and other observed spectral changes (a decrease in the band at 760 cm1 and shifts in the Fermi doublet components) have also been previously correlated in the literature with -, CH-, and cation- interactions. Finally, a subtle change in the Fermin doublet intensity ratio was also observed during the self-assembly process. The observed change is a well-established indicator that the hydrophobicity of the local environment in the indole ring is increasing. Taken together with the -system related markers, these findings suggest the formation of higher ordered Trp clusters mediated by - and CH- interactions, leading to the development of hydrophobic regions. We then extend the methodology to the study of the amyloid-like fibril formation of Trp in phosphate-buffered saline (PBS), a medium often considered inert in such studies. The results revealed markedly different and faster aggregation kinetics, suggesting the formation of structurally distinct aggregate structures in buffer compared to those obtained in pure water. Although additional studies are necessary to fully elucidate the effect of the buffer on the modulation of the aggregation pathways, some spectral changes suggest that, in its presence, Trp may be involved in cation- interactions that modulate the pi-stacking interactions during fibril formation. This could lead to tightly packed Trp indole groups within the supramolecular structure. This hypothesis is supported by the decrease in the intensity of the band at 760 cm1 , and the emergence of new bands at 722 and 734 cm1 . Similar spectral changes have been scarcely observed in the literature, but were correlated with interactions involving the -electron system. Additionally, other -interaction markers were also observed to change, such as shifts in the band at 1230 cm1 and in the Fermi doublet. Even though the interpretation of other spectral changes still requires refinement, our results demonstrate the capability and potential of Raman-based techniques to probe metabolite self-assembly at the molecular level under different physicochemical conditions.