An official website of the United States government
Steady-state fluorescence spectroscopy has a central role not only for sensing applications, but also in biophysics and imaging. Light switching probes, such as ruthenium dipyridophenazine complexes, have been used to study complex systems such as DNA, RNA, and amyloid fibrils. Nonetheless, steady-state spectroscopy is limited in the kind of information it can provide. In this paper, we use time-resolved spectroscopy for studying binding interactions between amyloid-β fibrillar structures and photoluminescent ligands. Using time-resolved spectroscopy, we demonstrate that ruthenium complexes with a pyrazino phenanthroline derivative can bind to two distinct binding sites on the surface of fibrillar amyloid-β, in contrast with previous studies using steady-state photoluminescence spectroscopy, which only identified one binding site for similar compounds. The second elusive binding site is revealed when deconvoluting the signals from the time-resolved decay traces, allowing the determination of dissociation constants of 3 and 2.2 μM. Molecular dynamic simulations agree with two binding sites on the surface of amyloid-β fibrils. Time-resolved spectroscopy was also used to monitor the aggregation of amyloid-β in real-time. In addition, we show that common polypyridine complexes can bind to amyloid-β also at two different binding sites. Information on how molecules bind to amyloid proteins is important to understand their toxicity and to design potential drugs that bind and quench their deleterious effects. The additional information contained in time-resolved spectroscopy provides a powerful tool not only for studying excited state dynamics but also for sensing and revealing important information about the system including hidden binding sites.
more »
« less
Michler’s hydrol blue elucidates structural differences in prion strains
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.
more »
« less
- Award ID(s):
- 1751174
- PAR ID:
- 10217214
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 117
- Issue:
- 47
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- 29677 to 29683
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Some proteins, including yeast translation termination factor Sup35 (eRF3) are capable of both stress-induced liquid-liquid phase separation (LLPS) and formation of solid fibrous aggregates (amyloids). Fragmentation and propagation of amyloid fibrils generates transmissible (in yeast, heritable) self-perpetuating protein agents, termed prions. Relationships between these processes are still poorly understood. Previous literature data suggested that the ability of Sup35 orthologs to form a prion is sporadically distributed in fungal evolution, and depends on amino acid composition of Sup35 prion domain (PrD), rather than on a evolutionarily variable specific sequence. We have studied two groups of proteins: 1) fungal Sup35 PrDs of various evolutionary origins, and 2) artificially synthesized “scrambled” variants of Saccharomyces cerevisiae Sup35 PrD, that possess identical amino acid composition but different sequences. These proteins were fused to fluorophores and expressed in S. cerevisiae cells. LLPS and amyloid/prion formation were assessed by fluorescence microscopy and biochemical approaches. Amino acid sequences were analyzed by various computational algorithms. Our data indicates that propagation of prion state strongly depends on the evolutionary distance from the host. In contrast, majority of proteins studied are capable of both LLPS and ability to form amyloid fibrils. These capabilities are associated with specific patterns of PrD amino acid distribution, that are broadly conserved among fungi. Notably, PrDs of different sequences differ from each other by their ability to convert from liquid condensates to amyloids, and relationship between these processes is apparently optimized in evolution. Moreover, heterotypic PrDs are can colocalize with each other within liquid condensates and influence amyloid conversion by each other. To conclude, LLPS and amyloid properties depend on specific evolutionarily conserved sequence patterns, indicating possible important biological roles for these processes. These patterns could potentially be used to predict LLPS and prion potential in other sequence contexts. This work was supported by NSF grant 2345660.more » « less
-
Long-term, super-resolution imaging of amyloid structures using transient amyloid binding microscopyAmyloid fibrils and tangles are signatures of Alzheimer disease, but nanometer-sized aggregation intermediates are hypothesized to be the structures most toxic to neurons. The structures of these oligomers are too small to be resolved by conventional light microscopy. We have developed a simple and versatile method, called transient amyloid binding (TAB), to image amyloid structures with nanoscale resolution using amyloidophilic dyes, such as Thioflavin T, without the need for covalent labeling or immunostaining of the amyloid protein. Transient binding of ThT molecules to amyloid structures over time generates photon bursts that are used to localize single fluorophores with nanometer precision. Continuous replenishment of fluorophores from the surrounding solution minimizes photobleaching, allowing us to visualize a single amyloid structure for hours to days. We show that TAB microscopy can image both the oligomeric and fibrillar stages of amyloid-β aggregation. We also demonstrate that TAB microscopy can image the structural remodeling of amyloid fibrils by epi-gallocatechin gallate. Finally, we utilize TAB imaging to observe the non-linear growth of amyloid fibrils.more » « less
-
The Y145Stop mutant of human prion protein (huPrP23-144) is associated with a familial prionopathy and provides a convenient in vitro model for investigating amyloid strains and cross-seeding barriers. huPrP23-144 fibrils feature a compact and relatively rigid parallel in-register β -sheet amyloid core spanning ∼30 C-terminal amino acid residues (∼112–141) and a large ∼90-residue dynamically disordered N-terminal tail domain. Here, we systematically evaluate the influence of this dynamic domain on the structure adopted by the huPrP23-144 amyloid core region, by investigating using magic-angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy a series of fibril samples formed by huPrP23-144 variants corresponding to deletions of large segments of the N-terminal tail. We find that deletion of the bulk of the N-terminal tail, up to residue 98, yields amyloid fibrils with native-like huPrP23-144 core structure. Interestingly, deletion of additional flexible residues in the stretch 99–106 located outside of the amyloid core yields shorter heterogenous fibrils with fingerprint NMR spectra that are clearly distinct from those for full-length huPrP23-144, suggestive of the onset of perturbations to the native structure and degree of molecular ordering for the core residues. For the deletion variant missing residues 99–106 we show that native huPrP23-144 core structure can be “restored” by seeding the fibril growth with preformed full-length huPrP23-144 fibrils.more » « less
-
Abstract Amyloid protein aggregation is associated with many neurodegenerative diseases, including amyloid‐β (Aβ)in Alzheimer disease, human islet amyloid polypeptide (hIAPP) in type II diabetes, and human calcitonin (hCT) in medullary thyroid carcinoma. Significant efforts have been made to develop different diagnostic and prevention strategies for the early detection and intervention of these disease‐causative protein aggregates. However, conventional design wisdoms are mostly limited to the molecules with either single function (amyloid imaging or amyloid prevention) or single targeting protein (Aβ, hIAPP, or hCT). Here, a rational design strategy of an amyloid‐aggregation‐induced emission (AIE)‐active molecule is demonstrated by conjugating an amyloid fragment of GNNQQNY (G7) with an AIE fluorescent molecule of triphenylvinyl benzoic acid (namely, G7‐TBA), making G7‐TBA as multiple‐target, dual‐function, amyloid probes and amyloid modulators for detecting, monitoring, and altering amyloid aggregation of three different amyloid proteins (Aβ, hIAPP, and hCT). G7‐TBA probe shows conformationally specific binding affinities to amyloid aggregates, switching from an “off” state (low fluorescence) for amyloid monomers to an “on” state (high fluorescence) for β‐structure‐rich amyloid oligomers and fibrils in aqueous solution. Further surface immobilization of TBA probes on surface plasmon resonance surfaces allows to amplify detection sensitivity and binding affinity to amyloid aggregates formed at different aggregation stages. G7‐TBA as amyloid modulator enables acceleration of amyloid fibrillization and selectively protects cells from hIAPP‐induced toxicity. The distinct amyloid detection and modulation of G7‐TBA are essentially derived from the cross‐seeding between G7 and amyloid aggregation via β‐structure interaction, which by far exceed the binding affinity between commercial ThT and amyloid aggregates. Such design concepts of amyloid‐AIE conjugates can be further explored as multiple‐function and target probes and/or modulators for biomedical applications.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2001732117
