The interaction of small molecules with the surface of amyloid assemblies is important for the detection and inhibition of amyloid formation. Thioflavin T (ThT), a small molecular rotor, has been used for the detection of amyloid fibrils for over half a century. The basis for detection is simple in that in the presence of fibrils the fluorescence of ThT is dramatically enhanced. The mechanism for this enhancement is not well understood but may depend on the determination of the conformation of ThT bound to the fibril surface. Here, we first use solution-state (1)H NMR to show that the on-off binding of ThT to the surface of insulin amyloid fibrils correlates with the enhancement of ThT fluorescence. We then show that the conformation of surface-bound ThT is twisted. The implications of this result in light of recent experimental and computational studies of the binding of ThT to amyloid or amyloid-like assemblies are discussed.

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The formation of amyloid fibrils is associated with incurable diseases including Alzheimer's, Parkinson's, and type 2 diabetes. Important mechanistic details of the self-assembly are unknown partly because of the absence of a clear structural characterization of intermediates. There is experimental evidence, however, for α-helical intermediates that has come primarily from circular dichroism spectroscopy. Here, we strengthen the evidence for helical intermediates by demonstrating helix-dipole effects in the early events of self-assembly. Previously, we showed that capped peptides containing the part of the islet amyloid polypeptide that may be responsible for the initial intermolecular contacts (Acetyl-R(11)LANFLVHSSNNFGA(25)-NH(2) and Acetyl-R(11)LANFLVHSGNNFGA(25)-NH(2) which contains the S20G mutation associated with early onset type 2 diabetes) self-assemble via helical intermediates [Liu et al. (2010) J. Am. Chem. Soc.132, 18223-18232]. We demonstrate here that when the peptides are uncapped, they do not self-assemble as indicated primarily by circular dichroism and nuclear magnetic resonance data. Self-assembly is restored when the charge on α-NH(3)(+) of Arg11 is eliminated but not when the charge on α-COO(-) of Ala25 is removed, consistent with the helicity of the peptides skewed toward the N-terminus. Our results strengthen the hypothesis that α-helical intermediates are on pathway to amyloid formation and indicate that the helix dipole is an attractive target for inhibiting the formation of α-helical assemblies.

Cationic amyloid fibrils found in human semen enhance the transmission of the human immunodeficiency virus (HIV) and thus, are named semen-derived enhancer of virus infection (SEVI). The mechanism for the enhancement of transmission is not completely understood but it has been proposed that SEVI neutralizes the repulsion that exists between the negatively charged viral envelope and host cell membrane. Consistent with this view, here we show that the fluorescence of cationic thioflavin T (ThT) in the presence of SEVI is weak, and thus ThT is not an efficient detector of SEVI. On the other hand, K114 ((trans, trans)-bromo-2,5-bis(4-hydroxystyryl)benzene) forms a highly fluorescent, phenolate-like species on the cationic surface of SEVI. This species does not form in the presence of amyloid fibrils from insulin and amyloid-b protein, both of which are efficiently detected by ThT fluorescence. Together, our results show that K114 is an efficient detector of SEVI.

A family of pyrazine-bridged, linear chain complexes of Cu(II) of the formula [CuL2(H2O)2(pz)](ClO4)2 [pz = pyrazine; L = n-methyl-2(1H)-pyridone, n = 3 (1), 5 (2), and 6 (3)] has been...