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Protein-induced photophysical changes to the amyloid indicator dye thioflavin T.

ABSTRACT: The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer's disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a ?-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural basis for binding specificity and for the changes to the photophysical properties of ThT remain poorly understood. Here, we report the co-crystal structures of ThT with two alternative states of ?-2 microglobulin (?2m); one monomeric, the other an amyloid-like oligomer. In the latter, the dye intercalates between ?-sheets orthogonal to the ?-strands. Importantly, the fluorophore is bound in such a manner that a photophysically relevant torsion is limited to a range of angles generally associated with low, not high, quantum yield. Quantum mechanical assessment of the fluorophore shows the electronic distribution to be strongly stabilized by aromatic interactions with the protein. Monomeric ?2m gives little increase in ThT fluorescence despite showing three fluorophores, at two binding sites, in configurations generally associated with high quantum yield. Our efforts fundamentally extend existing understanding about the origins of amyloid-induced photophysical changes. Specifically, the ?-sheet interface that characterizes amyloid acts both sterically and electronically to stabilize the fluorophore's ground state electronic distribution. By preventing the fluorophore from adopting its preferred excited state configuration, nonradiative relaxation pathways are minimized and quantum yield is increased.

SUBMITTER: Wolfe LS

PROVIDER: S-EPMC2947910 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Protein-induced photophysical changes to the amyloid indicator dye thioflavin T.

Wolfe Leslie S LS Calabrese Matthew F MF Nath Abhinav A Blaho Dorottya V DV Miranker Andrew D AD Xiong Yong Y

Proceedings of the National Academy of Sciences of the United States of America 20100908 39

The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer's disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a β-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural ba ...[more]

PMID: 20826442

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Project description:The persistence of high concentrations of beta-2-microglobulin (β2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of β2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length β2M (β2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6β2m and ΔN10β2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of β2M amyloid fibrils with affinity ~10⁴ M-1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with β2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-β2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10β2m fibrils from other β2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between β2m and ΔN6β2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the β2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit β2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

Dataset Information

Protein-induced photophysical changes to the amyloid indicator dye thioflavin T.

Publications

Protein-induced photophysical changes to the amyloid indicator dye thioflavin T.

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