Abstract
Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler’s hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 202001732 |
| Journal | Proceedings of the National Academy of Sciences |
| DOIs | |
| State | Published - Nov 10 2020 |
| Externally published | Yes |
Bibliographical note
KAUST Repository Item: Exported on 2020-11-16Acknowledged KAUST grant number(s): KUK-11-008-23
Acknowledgements: The late Professor Susan Lindquist was involved in the initiation of this project and the authors acknowledge (and miss) her scientific insight and enthusiasm. King Abdullah University of Science and Technology (Grant KUK-11-008-23) is gratefully acknowledged. We give
special thanks to Johan Johansson for assessing the purity of the Michler’s hydrol blue sample used in these experiments. Swedish National Infrastructure for Computing resources were used for the quantum mechanics calculations. This work was supported by grants from NSF (Grant 1751174), the Welch Foundation (Grant 1-1923-20170325), NIH (Grant NS-111236), the Cancer Prevention & Research Institute of Texas (Grant RR150076), the Lupe Murchison Foundation, and the Kinship Foundation (Searle Scholars Program) (all to K.K.F.). Support from the Hungarian Academy of Science within the Momentum Programme (Grant LP2016-2) is also acknowledged.
This publication acknowledges KAUST support, but has no KAUST affiliated authors.