Formation of amyloid fibrils underlies a wide range of human disorders, including Alzheimer's and prion diseases. The amyloid fibrils can be readily detected thanks to thioflavin T (ThT), a small molecule that gives strong fluorescence upon binding to amyloids. Using the amyloid fibrils of Aβ40 and Aβ42 involved in Alzheimer's disease, and of yeast prion protein Ure2, here we study three aspects of ThT binding to amyloids: quantification of amyloid fibrils using ThT, the optimal ThT concentration for monitoring amyloid formation and the effect of ThT on aggregation kinetics. We show that ThT fluorescence correlates linearly with amyloid concentration over ThT concentrations ranging from 0.2 to 500 µM. At a given amyloid concentration, the plot of ThT fluorescence versus ThT concentration exhibits a bell-shaped curve. The maximal fluorescence signal depends mostly on the total ThT concentration, rather than amyloid to ThT ratio. For the three proteins investigated, the maximal fluorescence is observed at ThT concentrations of 20–50 µM. Aggregation kinetics experiments in the presence of different ThT concentrations show that ThT has little effect on aggregation at concentrations of 20 µM or lower. ThT at concentrations of 50 µM or more could affect the shape of the aggregation curves, but this effect is protein-dependent and not universal.

Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.Keywords: site-directed spin labeling; EPR spectroscopy; side-chain conformation Supplemental material: see www.proteinscience.org Site-directed spin labeling (SDSL) has become a widely used tool to study protein structure, dynamics, and interactions (for reviews, see Hubbell et al. 1996Hubbell et al. , 1998Hubbell et al. , 2000Columbus and Hubbell 2002;Klug and Feix 2004;Fanucci and Cafiso 2006). The SDSL approach requires a cysteine residue to be introduced at a selected site and subsequently modified with a nitroxide reagent to generate a disulfidelinked nitroxide side chain. The most commonly employed spin-label side chain is designated R1 (Fig. 1). The EPR spectra of R1 directly encode information on the motion of the nitroxide, which in turn reflects structure and dynamics of the local protein environment at the labeling site. A primary goal in developing the SDSL method is to enable interpretation of EPR spectra in terms of protein structure and dynamics using well-characterized proteins as models. Success in this regard will allow SDSL to be used to infer these features in poorly characterized proteins that are challenging to other methods.At the present time, simple one-component EPR spectra that arise from R1 on solvent-exposed surfaces of a-helices and in ordered turns are reasonably well understood (Guo et al. 2007). At these sites, the motion of R1 is determined by the internal motion of the side chain and local backbone dynamics (Columbus et al. 2001), and the EPR spectra provide a means for mapping backbone dynamics (Columbus and Hubbell 2004 Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin-labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site-directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native-like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility.

A nitroxide side chain (R1) has been substituted at single sites along a helix-turn-helix motif in T4 lysozyme (residues 114-135). Together with previously published data, the new sites reported complete a continuous scan through the motif. Mutants with R1 at sites 115 and 118 were selected for crystallographic analysis to identify the structural origins of the corresponding two-component EPR spectra. At 115, R1 is shown to occupy two rotamers in the room temperature crystal structure, one of which has not been previously reported. The two components in the EPR spectrum apparently arise from differential interactions of the two rotamers with the surrounding structure, the most important of which is a hydrophobic interaction of the nitroxide ring. Interestingly, the crystal structure at 100 K reveals a single rotamer, emphasizing the possibility of rotamer selection in low-temperature crystal structures. Residue 118 is at a solvent-inaccessible site in the protein core, and the structure of 118R1, the first reported for the R1 side chain at a buried site, reveals how the side chain is accommodated in an overpacked core.

Deposition of Aβ in the brain is a pathological hallmark of Alzheimer's disease. There are two major isoforms of Aβ: the 42-residue Aβ42 and the 40-residue Aβ40. The only difference between Aβ42 and Aβ40 is that Aβ42 has two extra residues at the C-terminus. The amyloid plaques in Alzheimer's brains consist of mostly Aβ42 and some plaques contain only Aβ42, even though Aβ40 concentration is several-fold more than Aβ42. Using electron paramagnetic resonance, we studied the formation of amyloid fibrils using a mixture of Aβ42 and Aβ40 in vitro. We show that Aβ42 and Aβ40 form mixed fibrils in an interlaced manner, although Aβ40 is not as efficient as Aβ42 in terms of being incorporated into Aβ42 fibrils. Our results suggest that both Aβ42 and Aβ40 would be present in amyloid plaques if in vivo aggregation of Aβ were similar to the in vitro process. Therefore, there must be some mechanisms that lead to the preferential deposition of Aβ42 at the extracellular space. Identifying such mechanisms may open new avenues for therapeutic interventions to treat Alzheimer's disease.

Amyloid fibrils are associated with >20 fatal human disorders, including Alzheimer's, Parkinson's, and prion diseases. Knowledge of how soluble proteins assemble into amyloid fibrils remains elusive despite its potential usefulness for developing diagnostics and therapeutics. In at least some fibrils, runaway domain swapping has been proposed as a possible mechanism for fibril formation. In runaway domain swapping, each protein molecule swaps a domain into the complementary domain of the adjacent molecule along the fibril. Here we show that T7 endonuclease I, a naturally domain-swapped dimeric protein, can form amyloid-like fibrils. Using protein engineering, we designed a double-cysteine mutant that forms amyloid-like fibrils in which molecules of T7 endonuclease I are linked by intermolecular disulfide bonds. Because the disulfide bonds are designed to form only at the domain-swapped dimer interface, the resulting covalently linked fibrils show that T7 endonuclease I forms fibrils by a runaway domain swap. In addition, we show that the disulfide mutant exists in two conformations, only one of which is able to form fibrils. We also find that domain-swapped dimers, if locked in a close-ended dimeric form, are unable to form fibrils. Our study provides strong evidence for runaway domain swapping in the formation of an amyloid-like fibril and, consequently, a molecular explanation for specificity and stability of fibrils. In addition, our results suggest that inhibition of fibril formation for domain-swapped proteins may be achieved by stabilizing domain-swapped dimers.disulfide bond ͉ fibrillization ͉ protein aggregation ͉ protein design

Background: Soluble A␤42 oligomers, rather than insoluble amyloid fibrils, are toxic species in Alzheimer's disease. Results: We obtained structural restraints at all 42 residue positions in A␤42 oligomers and performed structural modeling. Conclusion: In oligomers, each A␤42 protein forms a single ␤-sheet with three antiparallel ␤-strands. Significance: Our novel structural model provides new structural framework for understanding oligomer-fibril interconversion and designing oligomer-targeted therapeutics.

Oligomerization of the 42-residue peptide Aβ42 plays a key role in the pathogenesis of Alzheimer disease. Despite great academic and medical interest, the structures of these oligomers have not been well characterized. Site-directed spin labeling combined with electron paramagnetic resonance spectroscopy is a powerful approach for studying structurally ill-defined systems, but its application in amyloid oligomer structure study has not been systematically explored. Here we report a comprehensive structural study on a toxic Aβ42 oligomer, called globulomer, using site-directed spin labeling complemented by other techniques. Transmission electron microscopy shows that these oligomers are globular structures with diameters of ∼7-8 nm. Circular dichroism shows primarily β-structures. X-ray powder diffraction suggests a highly ordered intrasheet hydrogen-bonding network and a heterogeneous intersheet packing. Residue-level mobility analysis on spin labels introduced at 14 different positions shows a structured state and a disordered state at all labeling sites. Side chain mobility analysis suggests that structural order increases from N- to C-terminal regions. Intermolecular distance measurements at 14 residue positions suggest that C-terminal residues Gly-29-Val-40 form a tightly packed core with intermolecular distances in a narrow range of 11.5-12.5 Å. These intermolecular distances rule out the existence of fibril-like parallel in-register β-structures and strongly suggest an antiparallel β-sheet arrangement in Aβ42 globulomers.