Binding to aggregated Aβ enhances ThT fluorescence
Thioflavin T is a benzothiazole-based dye (Figure 2A) first noted to bind amyloid by Vassar and Culling in 1959 (14) and later used in studying patient-derived amyloids by Naiki et al. (15–17). These groups noted that the fluorescence of ThT is quenched in solution, but that its quantum yield is greatly increased when bound to the β-sheet structure of amyloid fibrils (18). This same phenomenon was observed with synthetic Aβ fibrils (19), and ThT was adapted by LeVine (20) into a convenient, inexpensive assay for monitoring fibril formation in vitro. This protocol has been largely unchanged and is perhaps the most widely employed method for monitoring Aβ aggregation.
Figure 2. Thioflavin T (ThT) binds amyloids at multiple sites. (A) Chemical structures of ThT and a neutral analog, BTA-1, highlighting the major components: a benzothiazole ring system attached to functionalized aniline. The linker between these modules is shown in red. (B) Amyloids contain three distinct ThT binding sites, two with high density (BS1 and BS2) and one with low density (BS3). (C) Snapshots from molecular dynamics simulations illustrate ThT binding within two channels lined with either Phe residues (left) or both Phe and Val residues (right) (27,28). (D) A schematic model of the two ThT binding sites, illustrating their unique molecular features. The same color scheme is used in parts C and D.
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Thioflavin T fluorescence is thought to increase when bound to Aβ fibrils because of the changes in the rotational freedom of the carbon–carbon bond between the benzothiazole and aniline rings (Figure 2A) (18,21,22). In the unbound state, the ultrafast twisting dynamics around this bond are thought to cause rapid self-quenching of the excited state, resulting in low emission. However, upon binding to fibrils, the rotational freedom is apparently restricted and the excited state is readily populated. This concept was recently confirmed using a series of synthetic ThT analogs, which varied in their flexibility (22). The practical outcome of this mechanism is that ThT and its analogs can be used to spectroscopically quantify the amount of amyloid in a sample.
Binding modes of ThT to amyloid fibrils
The dramatic response of ThT’s fluorescence to amyloids suggests that a defined binding event takes place to restrict the motion of the compound. Thus, studying this interaction would be expected to reveal insights into the local amyloid topology and its molecular features. This strategy has been productive, with solid-state NMR (10), radiolabeling (23,24), competition assays (25), and molecular dynamics (MD) simulations (26–28) all having been used to evaluate ThT binding to amyloids. One of the first important observations was made by LeVine (25), who proposed that there are multiple binding sites for ThT on amyloids. Lockhart et al. (23) further refined this model using fluorescence and radiolabel-based assays. Together, these studies suggested the presence of three distinct binding sites (BS1, BS2, and BS3) on Aβ fibrils. Sites BS1 and BS2 are relatively abundant, with approximately one site per 4–35 Aβ monomers (Figure 2B). The less-abundant site, BS3 is found at approximately one site for every 300 monomers. Based on Forster resonance energy transfer measurements, binding sites BS1 and BS2 are thought to be in close proximity; however, occupancy was found to be neither cooperative nor competitive.
Further insights into how ThT binds in these different sites was supplied by MD simulations (27,28). Briefly, Wu et al. (27) simulated ThT binding to protofibrils composed of Aβ (16–22) and characterized the formation of three unique, populated clusters. Consistent with their earlier findings (28), the least populated binding cluster was located at the end of the protofibril in an orientation anti-parallel to the fibril axis. The two other clusters were more heavily populated and located parallel to the fibril axis (anti-parallel to the β-sheet). Together, these results seem to confirm Lockhart’s model of two high-density ThT sites (BS1 and BS2) and a single low-density site (BS3) (Figure 2B). In this model, BS1 and BS2 are composed of surface grooves created by aligned side chains in the fibril axis, which provide much of the binding energy. When bound in these grooves, MD simulations suggest that the ring systems adopt a planar organization, with the charged nitrogen exposed to solvent (26,29) (Figure 2C). Interestingly, similar grooves have been proposed in amyloid fibrils composed of many different proteins (e.g. α-synuclein and prions), which may explain why ThT fluorescence is also sensitive to other, unrelated amyloids. However, because different amyloid-forming peptides do not share a high sequence identity, these results also suggest that some degenerative feature(s), such as hydrophobicity or contacts with the peptide backbone, are responsible for ThT binding.
Consistent with this idea, a closer examination of the molecular models reveals interesting features of the two most populated ThT binding sites (Figure 2C) (27). These two binding channels are lined with at least five, spatially consecutive, hydrophobic (Phe only or Phe and Val) side chains, which are located on opposite ‘faces’ of the amyloid structure. Similar findings have been observed by Koide et al., who developed peptide self-assembly mimics that have repetitive, β-sheet amyloid-like structure (30–32). These soluble model proteins are amenable to crystallization, and co-crystals with bound ThT revealed that the compound binds in channels lined with five or six aromatic and hydrophobic side chains (31). Interestingly, it is critical that the favorable, hydrophobic residues within the channel are spatially consecutive, as two adjacent clusters of Tyr residues separated by a Lys and Glu showed no ThT binding (31). Collectively, these observations converge on a model in which five, aligned aromatic and/or hydrophobic residues are critical for ThT binding, while the exact identities of the side chains appear to be less important than their overall hydrophobicity.
An alternative binding mode was suggested by Groenning et al. using spectroscopy and molecular modeling to examine ThT binding to insulin fibrils. Although they confirmed that at least two distinct binding sites exist, with ThT binding predominantly parallel to the fibril axis (33,34), they further suggest that two ThT molecules in an excited-state dimer, or ‘excimer,’ form might bind to the grooves. Thus, a higher-order form of ThT, even as large as a micelle (35), might be involved in binding under some conditions and for some amyloids.
ThT also recognizes prefibrillar Aβ aggregates
As mentioned previously, prefibrillar intermediates are now thought to correlate with neurodegeneration, which has prompted interest in evaluating ThT binding to these structures. In the literature, there was initially debate over whether ThT binds oligomers and protofibrils. In fact, several groups initially defined ThT as a fibril-specific probe (36–39). However, methods for preparing prefibrillar structures have become more reliable, and comprehensive studies have now noted clear changes in fluorescence when ThT is added to prefibrils (40–42). For example, Walsh et al. (42) prepared samples of protofibrils and observed that they produce a concentration-dependent increase in ThT fluorescence. The binding of ThT to prefibrils is consistent with the binding information discussed earlier, as these structures are known to be rich in the hydrophobic, β-sheet content important for binding (42). For example, models of Aβ protofibrils have the requisite stretch of aligned hydrophobic residues one might expect to form the high-abundance ThT-binding site (43). However, fibrils and prefibrils are not identical in their binding to ThT, as many groups have noted that the maximum fluorescence induced by prefibrils is less (per mole of Aβ) than that stimulated by fibrils. For example, ThT fluorescence is modestly increased (1.5-fold) in the presence of Aβ oligomers (41) of either 1–40 or 1–42 Aβ (40), while fibrils often yield over 100-fold improvements in fluorescence (44). Moreover, using surface plasmon resonance (SPR), ThT (Kd = 498 nm) was shown to bind Aβ oligomers, but the total number of bound molecules was significantly less than in fibrils (40). These observations and others are likely consistent with other findings, because protofibrils are proposed to be more dynamic (45) and contain relatively fewer ThT-binding sites (46,47).
Interactions of ThT analogs with Aβ
Additional insights into the nature of the ThT-binding groove can be gained from studying synthetic ThT derivatives in which the molecular features of the molecule are systematically varied. In general, these derivatives are composed of a benzothiazole ring system attached to a substituted aniline (Figure 2A). Derivatives of this scaffold tend to have modifications at the amine of the aniline and at positions around the benzothiazole. Fortunately, many analogs have been explored as part of studies to develop imaging agents and; in many cases, the binding affinity of these compounds for amyloids has been reported. For example, Klunk et al. (48) synthesized several neutral ThT analogs, such as BTA-1, to explore the effect of the positive charge on the benzothiazole ring (Figure 2A). They found that each neutral analog bound better to Aβ than ThT, with the best having a 40-fold improved affinity. These findings suggest that the positive charge in ThT may be detrimental for binding, which is a model supported by MD simulations indicating that the neutral BTA-1 is able to bind deeper into the hydrophobic binding grooves (27). However, ThT does not entirely compete with BTA-1 for binding (25), and Lockhart et al. (23) observed different binding patterns between the two ligands. In silico data further reveal that the ring systems of BTA-1 are planar in the bound orientation, instead of in a slightly twisted orientation, as observed with the charged ThT scaffold (27). Collectively, these data support a model in which some of the ‘ThT binding sites’ (i.e. BS1, BS2 and BS3) are more favorable for ThT, while others prefer neutral ligands. Interestingly, removal of the positive charge does not seem to affect oligomer binding, suggesting that one of the binding sites may be more prevalent in prefibrils (49). One possibility is that some of the sites allow deeper binding grooves, perhaps permitting neutral ligands with increased access. This idea is supported by measurements of the dimensions of the two sites identified by modeling: one has an average depth (backbone to solvent) of 5.4 Å, while the other is 7.1 Å. This deeper site might allow better penetration of neutral derivatives and, consequently, more favorable buried surface area and better affinity. These sites are unique in other dimensions as well; the shallower site is wider (17 Å), while the deeper site is narrower (10 Å). It is not yet clear which site is BS1 or BS2, but these differences support the model that the two sites have distinct properties. To our knowledge, structure-guided design has not yet been used to rationally exploit these differences.
Synthetic ThT derivatives have also been useful in further refining the features of the binding sites. For example, the benzothiazole can be replaced by a roughly planar benzofuran, imidazo-pyridine, imidazole, or benzoxazole core, without impacting affinity (49–52). Additionally, the aniline may be replaced with other flat, rigid moieties (e.g. stilbene and cyanobenzyl) without significant consequence (53,54). Insertion of a planar styrene group between the benzothiazole and aniline is also well tolerated (53), while some substitutions, such as bithiophenes, even improve affinity (55). In addition, these substitutions need not be aromatic, because methyl-piperazine groups appended to the aniline are tolerated (56). Interestingly, some of these substitutions extend the end-to-end distance of the ThT-like molecule by over 25%, suggesting that the binding channel can accommodate relatively long molecules, as long as they are planar and hydrophobic. However, there are limitations to the size of this channel, because installation of a large, freely rotatable rhenium chelate to the aniline abolishes binding (57). Appending the same group to the opposite end of the molecule actually enhanced binding by eightfold, which suggests that the dimensions of the channel are limited in some regions.
More subtle substitutions to the benzothiazole and aniline groups also help define the nature of the ThT binding site(s). For instance, the di-methyl amine group can be moved to the benzothiazole on the opposite side of the molecule without any consequence to binding affinity (58), suggesting that ThT derivatives may be able to bind in either orientation (Figure 2D). However, small alkyl-type substitutions in key positions appear to impact the binding mode (59). For example, a methyl group at the six-position of the benzothiazole ring is favored only if no methyl substitutions are present on the amine of the aniline (Ki = 9.5 nm). Alternatively, two methyl groups on this amine were favored if the six-position was hydrogen (Ki = 4 nm). Interestingly, a more polar, hydroxyl at the six-position was tolerated only if the aniline was substituted with at least one methyl group, suggesting that overall hydrophobicity is a critical element for binding.
In summary, multiple experiments have converged on a model in which amyloids contain up to three different binding sites. The two major sites (BS1 and BS2) are parallel to the fibril axis and are sensitive to relatively modest increases in steric size in some positions, while they can be readily elongated in the channel if overall planarity is maintained (Figure 2D). The major contacts with ThT are through aromatic and/or hydrophobic side chains in the parallel groove, and neutral derivatives bind tighter than their charged counterparts. It is important to note that BS1 and BS2 are present in roughly equivalent numbers (at least on fibrils) and that the experiments discussed here often focus on the composite affinities. Differences in the way that ThT derivatives bind the different sites might easily be masked in these studies and, moreover, little is likely learned about the requirements at BS3 in these types of experiments.