Kinetic Transition in Amyloid Assembly As a Screening Assay for Oligomer-Selective Dyes

Article Kinetic Transition in Amyloid Assembly as a Screening Assay for Oligomer-Selective Dyes

Jeremy Barton, D. Sebastian Arias, Chamani Niyangoda, Gustavo Borjas, Nathan Le, Saefallah Mohamed and Martin Muschol * Department of Physics, University of South Florida, Tampa, FL 33620, USA; [email protected] (J.B.); [email protected] (D.S.A.); [email protected] (C.N.); [email protected] (G.B.); [email protected] (N.L.); [email protected] (S.M.) * Correspondence: [email protected]; Tel.: +1-813-974-2564

Received: 6 September 2019; Accepted: 25 September 2019; Published: 27 September 2019

Abstract: Assembly of amyloid fibrils and small globular oligomers is associated with a significant number of human disorders that include Alzheimer’s disease, senile systemic amyloidosis, and type II diabetes. Recent findings implicate small amyloid oligomers as the dominant aggregate species mediating the toxic effects in these disorders. However, validation of this hypothesis has been hampered by the dearth of experimental techniques to detect, quantify, and discriminate oligomeric intermediates from late-stage fibrils, in vitro and in vivo. We have shown that the onset of significant oligomer formation is associated with a transition in thioflavin T kinetics from sigmoidal to biphasic kinetics. Here we showed that this transition can be exploited for screening fluorophores for preferential responses to oligomer over fibril formation. This assay identified crystal violet as a strongly selective oligomer-indicator dye for lysozyme. Simultaneous recordings of amyloid kinetics with thioflavin T and crystal violet enabled us to separate the combined signals into their underlying oligomeric and fibrillar components. We provided further evidence that this screening assay could be extended to amyloid-β peptides under physiological conditions. Identification of oligomer-selective dyes not only holds the promise of biomedical applications but provides new approaches for unraveling the mechanisms underlying oligomer versus fibril formation in amyloid assembly.

Keywords: oligomer formation; amyloidosis; assembly pathway; dye ﬂuorescence; thioﬂavin T

1. Introduction Deposits of amyloid protein fibrils with their characteristic cross-β sheet architecture are the dominant pathological feature associated with a wide variety of age-related human disorders [1–7]. Most prominent among these age-related amyloid diseases are Alzheimer’s disease and type-II diabetes [8–11]. Accumulating evidence has implicated transient populations of small globular oligomers (gOs), first discovered during in vitro growth of amyloid proteins, as perhaps the most dominant contributors to the clinical symptoms of amyloid diseases [12–20]. One of the major obstacles towards elucidating whether and how amyloid oligomer and fibril formation are linked and in ascertaining how oligomers affect the etiology of disease symptoms is the limited range of techniques for detecting and discriminating oligomers from concurrent amyloid fibrils. Current techniques for detecting and characterizing amyloid oligomers include atomic force microscopy (AFM) [21–23], photo-induced, cross-linking of unmodified proteins (PICUP) [24], mass spectrometry [25], optical spectroscopy [26,27] and, most widely used, immunostaining with conformation-dependent anti-oligomer antibodies [28,29]. However, with the exception of anti-oligomer antibodies, none of the aforementioned techniques lend themselves for oligomer detection ex vivo, let alone in vivo. In addition, they all suffer from poor temporal resolution, which is a serious obstacle for kinetics studies to reveal the role of oligomers in fibril formation. These

Biomolecules 2019, 9, 539; doi:10.3390/biom9100539 www.mdpi.com/journal/biomolecules Biomolecules 2019, 9, 539 2 of 15 limitations on detecting, quantifying, and monitoring oligomers, combined with the intrinsically transient character of amyloid oligomers, has hampered efforts at unravelling the basic mechanisms underlying oligomer formation, at elucidating their mechanisms of cell toxicity, or at correlating disease symptoms with the accumulations of oligomers in vivo. Yet, these are critical steps towards establishing the role of oligomers in disease etiology and for developing targeted therapeutic interventions. Small molecules with a high selectivity for detecting amyloid oligomers over late-stage fibrils would be particularly useful. The benefits of small fluorophores, in particular, is highlighted by the commonly used amyloid indicator thioflavin T (ThT), with its high fluorescence enhancement in the presence of amyloid fibril [27,30,31]. It offers superior temporal resolution and sensitivity for monitoring fibril growth kinetics, which has been critical to the development of molecular models of amyloid fibril nucleation and growth [30,32,33]. Its membrane-permeant variant thioflavin S, in turn, is a commonly used histological amyloid stain [34,35]. ThT has also served as a template for the amyloid-binding moiety of the first positron emission tomography (PET) imaging probe Pittsburgh compound B [36]. However, ThT’s fluorescence response and binding affinity are strongly skewed towards late-stage fibrils over oligomers [27]. Hence, identification of novel oligomer-selective dyes (OSDs) would offer an important new modality for monitoring amyloid oligomer formation in vitro and for developing probes for oligomer detection in vivo [37]. Several such dyes have been tested and developed, often using fluorescence spectroscopy correlated with high-resolution imaging to establish oligomer selectivity [38–40]. However, so far they have found limited acceptance—partially due to the need for custom synthesis, the lack of commercial availability, or limited applicability. Ideally one would like a straightforward and reliable assay for screening a large set of dyes for select responses to amyloid oligomers over fibrils. Such efforts are hampered by the challenge of generating oligomer preparations not contaminated by fibril admixtures, and of stabilizing the intrinsically transient and metastable oligomers. Here, we investigate the use of a transition in amyloid kinetics induced by oligomer formation, which circumvents the need for isolating amyloid oligomers, to screen for oligomer-selective dyes (OSD). Specifically, we found that the formation of metastable globular oligomers is accompanied by the onset of biphasic growth kinetics [41]. We have previously shown that amyloid assembly of hen egg-white lysozyme (hewL) and of a dimeric Aβ40 construct (dimAβ), as recorded with the indicator dye thioflavin T (ThT), undergo a sharp transition from essentially oligomer-free sigmoidal to oligomer-dominated biphasic kinetics, upon crossing a (protein and salt concentration dependent) threshold [41,42]. Figure1a shows sigmoidal kinetics recorded with ThT for hewL under the amyloid growth conditions used for subsequent experiments. An extended lag period, indicated by a complete lack of a ThT increase (notice semi-log scale) is followed by a rapidly accelerating upswing and plateau. AFM images document the absence of any discernible populations of prefibrillar nuclei, during the lag periods (Figure1b, 24 h), with increasing numbers of progressively longer rigid fibrils emerging during the rapid upswing in ThT (Figure1b, 94 h). Figure1c displays an example of well-developed biphasic ThT kinetics emerging at higher hewL concentrations. Then, ThT fluorescence increases immediately and reaches an initial plateau, which is followed by a second upswing and plateau. This switch in assembly kinetics is associated with a dramatic change in observable aggregate morphologies. The initial phase of biphasic growth yields significant quantities of globular oligomers (gOs), which subsequently polymerize into curvilinear fibrils (CFs) (Figure1D, 24 & 94 h). Rigid fibrils (RFs) only gradually emerge following the second upswing in ThT, with gO/CFs persisting long into the second plateau phase. (Figure1D, 94 & 144 h). Biomolecules 2019, 9, x FOR PEER REVIEW 3 of 15

nucleation rates and by buffering monomers. The gO/CF-induced retardation of RF formation is discernible by comparing the ThT amplitudes at the end of the biphasic (Figure 1a) versus sigmoidal (Figure 1c) growth. The ThT amplitude of the high concentration biphasic solution, at the end of the incubation period, reached only about 1/2 the ThT amplitude for the low-concentration sigmoidal solution. In addition, the RFs emerging during the biphasic growth were decorated by gO/CFs. We have argued that it is this interactions between RFs and gO/CFs that retards RF secondary nucleation [41]. For the purpose of the subsequent dye screening assay, though, we only relied on the fact that sigmoidal kinetics indicates essentially oligomer-free RF formation, while the initial phase of biphasic growth is dominated by gO/CF formation. To detect oligomer-specific dye responses, we identified Biomoleculesgrowth conditions2019, 9, 539 for which the oligomer-dominated phase of biphasic growth developed a plateau,3 of 15 separated in time from subsequent fibril nucleation and growth (see Figure 1b).

24 h 94 h (a) (c)

24 h 94 h 144 h (b) (d)

FigureFigure 1. 1.Sigmoidal Sigmoidal versus versus biphasicbiphasic amyloidamyloid kinetics and and corresponding corresponding aggregate aggregate morphologies. morphologies. AmyloidAmyloid growth growth kinetics kinetics ofof henhen egg-whiteegg-white lysozymelysozyme (hewL), (hewL), recorded recorded with with thioflavin thioﬂavin T (ThT), T (ThT), displayingdisplaying either either ( a()a) sigmoidal sigmoidal (21(21 µμMM hewL)hewL) or ( bb)) biphasic biphasic (350 (350 μµMM hewL) hewL) growth growth kinetics kinetics (pH (pH 2, 2, 52 °C, 400 mM NaCl). Second panel in (a) is the same data on a semi-log scale to highlight the flat 52 ◦C, 400 mM NaCl). Second panel in (a) is the same data on a semi-log scale to highlight the ﬂat baseline. Spikes in the traces mark the times when aliquots were collected for imaging. Atomic force baseline. Spikes in the traces mark the times when aliquots were collected for imaging. Atomic force microscopy (AFM) images of the aggregate morphologies observed during (c) sigmoidal versus (d) microscopy (AFM) images of the aggregate morphologies observed during (c) sigmoidal versus (d) biphasic growth of hewL, at the indicated time points. False color height scale: 3 nm. biphasic growth of hewL, at the indicated time points. False color height scale: 3 nm.

WeWe have have provided multiple evidencepieces of thatevidence this transitionto indicate isthat due the to gO/CFs a micelle-like emerging concentration during the biphasic threshold growth of either hewL or dimAβ match the properties of toxic oligomers associated with for the gO/CFs formation which we called “critical oligomer concentration”. This matches with prior amyloidoses. First and foremost, gO/CFs are early-stage metastable aggregates which become reports of a “critical micelle concentration” (or CMC) for oligomer formation in Aβ42 [43,44] and eventually superseded by the ‘traditional’ late-stage RFs [41,42]. Their metastability is highlighted by with the biphasic kinetics associated with the formation of long-lived oligomers in Aβ40 [41,42,45]. the initial plateau phase seen in the ThT traces (see Figure 1b) and their prolonged persistence, even In addition, we confirmed that these gO/CFs were metastable. Intriguingly, they also retarded the during the late stages of RF growth. The gO/CFs also display the well-defined globular morphology kineticsof toxic of oligomers subsequent which, RF nucleation in the case and of growth,hewL, are presumably composed byof reducingabout eight secondary monomers fibril [23]. nucleation These ratesgOs and subsequently by buffering polymerize monomers. to form The gOCFs,/CF-induced with cross-sections retardation identical of RF to formation those of their is discernible octameric by comparingbuilding blocks, the ThT and amplitudes distinct from at thethe endmonomeric of the biphasic cross-section (Figure of1 thea) versusRF filaments sigmoidal [23,46]. (Figure Again,1c) growth.CFs are The structures ThT amplitude frequently of theassociated high concentration with toxic oligomeric biphasic solution, preparations. at the Both end ofCD the and incubation FTIR period,further reached indicate only that aboutthese early-stage 1/2 the ThT gO/CFs amplitude share the for spectroscopic the low-concentration signatures sigmoidalof amyloids. solution. In FTIR, In addition,gO/CFs thedisplay RFs emerginga prominent during peak thein the biphasic Amide growthI “amyloid were band” decorated between by 1610 gO/CFs. and 1630 We have cm−1 argued[47]. thatYet it the is thispeak interactions wavenumber between is distinct RFs from and RFs gO/ CFsand thattypically retards shows RF secondaryadditional absorption nucleation around [41]. For the1690 purpose cm−1, ofwhich the subsequentis considered dye indicative screening of assay,antiparallel though, β-sheet we only structures relied [26,27]. on the factFTIR that spectra sigmoidal for kineticshewL indicatesRF and gO/CFs essentially used oligomer-freein our experiments RF formation, are shown while in Figure the initial 2. They phase match of biphasic our previous growth is dominated by gO/CF formation. To detect oligomer-specific dye responses, we identified growth conditions for which the oligomer-dominated phase of biphasic growth developed a plateau, separated in time from subsequent fibril nucleation and growth (see Figure1b). We have multiple pieces of evidence to indicate that the gO/CFs emerging during the biphasic growth of either hewL or dimAβ match the properties of toxic oligomers associated with amyloidoses. First and foremost, gO/CFs are early-stage metastable aggregates which become eventually superseded by the ‘traditional’ late-stage RFs [41,42]. Their metastability is highlighted by the initial plateau phase seen in the ThT traces (see Figure1b) and their prolonged persistence, even during the late stages of RF growth. The gO/CFs also display the well-defined globular morphology of toxic oligomers which, in the case of hewL, are composed of about eight monomers [23]. These gOs subsequently polymerize to form CFs, with cross-sections identical to those of their octameric building blocks, and distinct from the monomeric cross-section of the RF filaments [23,46]. Again, CFs are structures Biomolecules 2019, 9, 539 4 of 15 frequently associated with toxic oligomeric preparations. Both CD and FTIR further indicate that these early-stage gO/CFs share the spectroscopic signatures of amyloids. In FTIR, gO/CFs display 1 a prominent peak in the Amide I “amyloid band” between 1610 and 1630 cm− [47]. Yet the peak 1 wavenumber is distinct from RFs and typically shows additional absorption around 1690 cm− , which is consideredBiomolecules 2019 indicative, 9, x FOR PEER of antiparallel REVIEW β-sheet structures [26,27]. FTIR spectra for hewL RF and4 gO of /15CFs used in our experiments are shown in Figure2. They match our previous observations and replicate observations and replicate spectroscopic features of Aβ oligomers and their proposed 3D structure spectroscopic features of Aβ oligomers and their proposed 3D structure [26,27,48]. Similarly, the [26,27,48]. Similarly, the noticeable but muted ThT amplitudes associated with gO/CF formation, noticeable but muted ThT amplitudes associated with gO/CF formation, particularly when compared particularly when compared to equivalent concentrations of RFs, matches the weak ThT responses to to equivalent concentrations of RFs, matches the weak ThT responses to gO/CFs for a wide range of gO/CFs for a wide range of oligomeric amyloid species [23,27,41,42,46]. Oligomers of hewL have also oligomericbeen shown amyloid to react species with oligomer-specific [23,27,41,42,46]. antibodi Oligomerses and of hewLinduce have neurodegeneration, also been shown when to reactinjected with oligomer-specificinto rat brains [49]. antibodies Hence,and the inducegO/CFsneurodegeneration, emerging during the when initial injected phase of into biphasic rat brains growth [49]. share Hence, thebasic gO/ CFscharacteristics emerging duringof toxic theoligomers initial phaseseen with of biphasic various amyloid-forming growth share basic proteins characteristics and peptides of toxic— oligomerseven if the seen specific with in various vitro conditions amyloid-forming used to generate proteins them and during peptides—even the biphasic growth if the specific do not matchin vitro conditionsthose present used in to vivo. generate It is worth them noting during that the the biphasic initial growthdiscovery do of not toxic match oligomers, those presentsubsequentin vivo assays. It is worthfor their noting generation, that the initial the biophysical discovery characterization, of toxic oligomers, as subsequentwell as the development assays for their of anti-oligomer generation, the biophysicalantibodies, characterization, similarly, relied asonwell in vitro as the growth development of oligomers of anti-oligomer under various, antibodies, often non-physiological similarly, relied ongrowth in vitro conditions. growth of oligomers under various, often non-physiological growth conditions.

(a) (b)

Figure 2. FTIR spectra of hewL monomers, curvilinear fibrils (CFs) and Rigid fibrils (RFs). (a) Peak-matched FTIR absorption spectra of the Amide-I band for hewL monomers and isolated CFs andFigure RFs at 2. pH FTIR 2. CFsspectra and of RFs hewL show monomers, a prominent curvilinear peak in fibrils the characteristic (CFs) and Rigid amyloid fibrils band (RFs). between (a) Peak- 1630 matched FTIR1 absorption spectra of the Amide-I band for hewL monomers and isolated CFs and RFs and 1610 cm− . Yet their peak wavenumbers are slightly but reproducibly shifted with respect to each at pH 2. CFs and RFs show a prominent peak in the characteristic amyloid band between 1630 and other. (b) The CF and RF differences spectra obtained after subtraction of the monomer spectrum in (a). 1610 cm−1. Yet their peak wavenumbers are slightly but reproducibly shifted with respect to each Here,other. we (b evaluated) The CF and the RF responses differences ofspectra fluorescent obtained dyes after tosubtraction RF-dominated of the monomer sigmoidal spectrum versus in gO/CF dominated(a). biphasic growth, as an assay to screen for oligomer-selective dyes (OSDs). Specifically, we used sigmoidal versus biphasic growth conditions of hen egg–white lysozyme, during amyloid Here, we evaluated the responses of fluorescent dyes to RF-dominated sigmoidal versus gO/CF assemblydominated at pH biphasic 2 (Figure growth,1), to investigateas an assay theto screen oligomer-selectivity for oligomer-selective for a small dyes set (OSDs). of fluorescent Specifically, dyes. Forwe the used most sigmoidal promising versus candidate, biphasic we growth identified conditions the triarylmethane of hen egg–white dye crystal lysozyme, violet during (CV), weamyloid further investigatedassembly at whether pH 2 (Figure its apparent 1), to investigate oligomer the selectivity oligomer-selectivity instead arose for a small from set interference of fluorescent with dyes. fibril assembly.For the Wemost next promising determined candidate, whether we CVidentified could bethe used triarylmethane for simultaneous dye crystal recordings violet of(CV), amyloid we kineticsfurther with investigated ThT and, whether therefore, its forapparent reconstructing oligomer theselectivity separated instead contributions arose from of interference oligomers with versus fibrilsfibril to assembly. the compound We next dye kinetics.determined To expandwhether our CV approach, could be weused established for simultaneous that the tworecordings Alzheimer’s of diseaseamyloid peptides, kinetics A withβ40 ThT and and, Aβ42, therefore, undergo for a reconstructing similar kinetic the transition separated under contributions physiological of oligomers growth conditions.versus fibrils This to indicates the compound that the dye proposed kinetics. screening To expand approach, our approach, tested herewe established for dye responses that the duringtwo amyloidAlzheimer’s formation disease of a non-humanpeptides, A proteinβ40 and under Aβ42, non-physiological undergo a similar growth kinetic conditions, transition can beunder readily extendedphysiological to screen growth for OSDs conditions. for the This two indicates main peptides that the implicatedproposed screening in Alzheimer’s approach, disease. tested here for dye responses during amyloid formation of a non-human protein under non-physiological growth conditions, can be readily extended to screen for OSDs for the two main peptides implicated in Alzheimer’s disease.

2. Materials and Methods

2.1. Protein and Chemicals Biomolecules 2019, 9, 539 5 of 15

2. Materials and Methods

2.1. Protein and Chemicals Two times recrystallized, dialyzed, and lyophilized hen egg white lysozyme (hewL) was purchased from Worthington Biochemicals (Lakewood, NJ, USA) and Aβ40 was purchased from rPeptide (Watkinsville, GA, USA). Ultrapure grade Thioﬂavin T (ThT) was obtained from Anaspec (Freemont, CA, USA). Crystal violet (CV) and other chemicals were from Fisher Scientiﬁc (Pittsburgh, PA, USA) and were of reagent grade or better. All solutions were prepared using 18 MΩ water from a reverse osmosis unit (Barnstead E-pure, Dubuque, IA, USA).

2.2. Preparation of HewL, Aβ40, Aβ42, CV, and ThT Solutions

Lyophilized hewL was dissolved at twice its final concentration in a 25 mM KH2PO4 pH 2 buffer, with any residual aggregates dissolved by incubating the solution for a few minutes at 42 ◦C. [50] Samples were typically filtered through 220 nm PVDF (Fisherbrand, Fisher Scientific, Pittsburgh, PA, USA) and 50-nm PES (Tisch Scientific, North Bend, OH, USA) pore size syringe filters. The resulting solutions were highly monodisperse, as assessed with both dynamic light scattering and SDS–PAGE [50]. A series of hewL solutions at different concentrations was generated by diluting this hewL/buffer stock with buffer, to twice its final hewL concentration and then mixing it 1:1, with a NaCl/25 mM KH2PO4 pH 2 stock solution at twice the final salt concentrations. Actual lysozyme 1 concentrations were determined from UV absorption measurements at 280 nm (ε280 = 2.64 mL mg− 1 cm− ). Solutions for a series of hewL (or NaCl) concentrations spanning the transition from sigmoidal to biphasic behavior were prepared, and triplicates of each solutions were placed in 96-well glass bottom plates and were sealed. Lyophilized Aβ40 or Aβ42 peptide was dissolved in 100 mM NaOH and injected into a Superdex 75 10/300 GL column on an fast protein liquid chromatography (FPLC) system (Äkta Pure, GE, USA), using a 35 mM Na2HPO4 running buffer at pH 11. The monomer fraction was collected and kept on ice. The resulting Aβ monomer concentrations were measured using optical absorption at 280 nm with ε = (1470 20) M 1 cm 1 [51,52]. This Aβ stock was diluted 280 ± − − into ice-cold 35 mM Na2HPO4 at pH 11 at the highest Aβ concentration and the pH was adjusted to pH 7.4, by addition of 1.5% (by vol.) of 1M NaH2PO4. CV and ThT stock solutions were prepared at a concentration of 1 mM dye in distilled water and passed through 220 nm syringe filters. Actual dye stock concentrations were determined from 1 1 1 1 absorption using ε590 = 87,000 M− cm− for CV and ε412 = 28,800 M− cm− for ThT, respectively. ThT and CV were added to protein solutions, either separately or together, as indicated. Unless indicated otherwise, ThT concentrations were 15 µM and CV concentrations were 5 µM.

2.3. Measuring Amyloid Growth Kinetics with Thioflavin T and Crystal Violet Using a Fluostar Omega plate reader (BMG Labtech, Offenburg, Germany), hewL or Aβ 40 amyloid growth kinetics were measured during incubation at 52 ◦C (hewL) or 27 ◦C (Aβ 40), respectively. CV fluorescence was excited using built-in bandpass filters centered at 544 or 584 nm, respectively, with emission measured with a 620 nm bandpass filter. ThT fluorescence was excited with a 448 nm bandpass filter and emission was collected using a 482 nm bandpass filter.

2.4. Atomic Force Microscopy Amyloid aggregates were imaged in air with a MFP-3D atomic-force microscope (Asylum Research, Santa Barbara, CA, USA) using NSC36/NoAl (Mikromasch, San Jose, CA, USA) or PFP-FMR-50 (Nanosensor, Neuchatel, Switzerland) silicon tips with a nominal tip radii of 10 nm and 7 nm, respectively. The cantilever had a typical spring constant and resonance frequency of 2 nN/nm and 70 kHz, respectively. It was driven at 60–70 kHz in alternating current mode and at a scan rate of 0.5 Hz, acquiring images at 512 512 pixel resolution. Raw image data were corrected for image bow × and slope. For imaging, 75 µL of the sample solution was typically diluted 20- to 100-fold into the same Biomolecules 2019, 9, 539 6 of 15 salt/buffer solution used during growth, deposited onto freshly cleaved mica for 3–5 min, rinsed with deionized water, and dried with dry nitrogen. Amplitude, phase, and height images were collected for the same sample area. False-color heights were subsequently superimposed over either amplitude or phase images, offline. Biomolecules 2019, 9, x FOR PEER REVIEW 6 of 15 2.5. Transmission Electron Microscopy Amyloid2.5. Transmission samples Electron were dilutedMicroscopy into distilled water and 5 µL of the diluted sample was placed on FormvarAmyloid/carbon-film-coated, samples were diluted 200 mesh into distilled copper water grids and and 5 wasμL of allowed the diluted to airsample dry. was A 5 placedµL aliquot of distilledon Formvar/carbon-film-coated water was added to the, 200 grid mesh and copper blotted grids 3 times, and was to allowed dissolve to salt air dry. crystals. A 5 μL The aliquot grids of were negativelydistilled stained water withwas added 5 µL of to 8% the (w grid/v) uranyland blotted acetate, 3 times, for 1 to min, dissolve and were salt crystals. then blotted. The grids Excess were uranyl acetatenegatively was removed stained by with repeated 5 μL of washing 8% (w/v)with uranyl distilled acetate, water for 1 min, and theand gridswere werethen blotted. left to airExcess dry. The uranyl acetate was removed by repeated washing with distilled water and the grids were left to air grids were imaged using an FEI Morgani transmission electron microscope at 60 kV with an Olympus dry. The grids were imaged using an FEI Morgani transmission electron microscope at 60 kV with an MegaView III camera (Center Valley, PA, USA). Olympus MegaView III camera (Center Valley, PA, USA). 2.6. Fourier-Transform Infrared Spectroscopy (FTIR) 2.6. Fourier-Transform Infrared Spectroscopy (FTIR) Aggregates were prepared by growing them in a heat block at 52 ◦C. To remove monomer contributionsAggregates to FTIR were spectra, prepared aggregate by growing solutions them in were a heat centrifuged block at 52 in°C. 2 To mL remove centrifuge monomer tubes at 15,000contributionsg overnight. to FTIR The resultingspectra, aggregate aggregate solution pellet wass were dissolved centrifuged in a in bu 2ff ermL solution centrifuge and tubes the processat × was repeated15,000g overnight. 3–4 times. The Removal resultingof aggregate monomers pellet was was assessed dissolved using in a buffer dynamic solution light and scattering the process and UV was repeated 3–4 times. Removal of monomers was assessed using dynamic light scattering and UV absorption of the supernatant. For the FTIR spectra, 30 µL of the samples were deposited on the silicon absorption of the supernatant. For the FTIR spectra, 30 μL of the samples were deposited on the crystalsilicon of a crystal Bruker of Optik a Bruker Vertex Optik 70 Vertex (Ettlingen, 70 (Ettli Germany)ngen, Germany) spectrometer. spectrometer. Baseline Baseline scans scans of of the the bu ffer 1 solutionbuffer were solution taken were using taken 1000 using scans 1000 at scans 2 cm −at 2resolution, cm−1 resolution, and sampleand sample spectra spectra were were acquired acquired using 1 700 scansusing over700 ascans range over from a 3000range tofrom 1000 3000 cm− towavenumbers. 1000 cm−1 wavenumbers. Raw FTIR Raw spectra FTIR were spectra normalized were to the peaknormalized value of to their the peak Amide-I value band. of their Diff Amide-Ierence spectra band. Difference of RFs and spectra CFs had of theRFs normalizedand CFs had monomer the spectrumnormalized subtracted. monomer spectrum subtracted.

3. Results3. Results We monitoredWe monitored a small, a small, select select set set of of dyes dyes forfor theirtheir fluorescence fluorescence responses responses to either to either pure pure sigmoidal sigmoidal or well-definedor well-defined biphasic biphasic growth growth conditions. conditions. The dy dyeses chosen chosen for for this this limited limited screen screen were were either either knownknown amyloid amyloid dyes, dyes, molecular molecular rotors, rotors, hydrophobichydrophobic dyes, dyes, or or dyes dyes closely closely associated associated with withour our currentcurrent lead lead compound—crystal compound—crystal violet. violet. The The structurestructure of of the the dyes dyes we we found found to display to display some some level levelof of oligomer-selectivity,oligomer-selectivity, together together with with the the reference reference dye dye thioflavinthioflavin T, T, are are shown shown in inFigure Figure 3. 3.

X-34 acridine orange acid fuchsin crystal violet thioflavin T

FigureFigure 3. Summary 3. Summary of dyeof dye structures. structures. ChemicalChemical structure structure of of dyes dyes in inthis this study study with with indications indications of of oligomeroligomer selectivity, selectivity, together together with with the the reference reference amyloid amyloid indicator dye dye Thioflavin Thioﬂavin T. T.

To induceTo induce the the transition transition from from sigmoidal sigmoidal toto biphasicbiphasic behavior, behavior, we we either either used used different different salt salt concentrationsconcentrations while while fixing fixing the the hewL hewL concentration, concentration, or or vice vice versa. versa. Figure Figure 4 compares4 compares the thesigmoidal sigmoidal versusversus the biphasic the biphasic responses responses of of ThT ThT to to the the responses responses recorded recorded with with the the amyloid amyloid dye dye X-34, X-34, as well as wellas as with acridinewith acridine orange orange and and acid acid fuchsin. fuchsin. Each Each ofof thesethese dyes dyes showed showed some some sign sign of selectivity of selectivity for gO/CFs for gO /CFs over RFs. However, either their fluorescence responses were weak (acridine orange) or their over RFs. However, either their fluorescence responses were weak (acridine orange) or their selectivity selectivity for gO/CFs over RFs were quite modest (acid fuchsin), they displayed excessive bleaching for gO(X-34)/CFs or over had RFs very were high quite levels modest of baseline (acid fluorescence fuchsin), they(X-34, displayed acridine orange). excessive Screening bleaching a rather (X-34) or had verylimited high set levelsof dyesof did, baseline however, fluorescence also yield the (X-34, triarylmethane acridine dye orange). crystal Screening violet (CV) a and rather the closely limited set of dyesrelated did, methyl however, violet also as promising yield the candidates triarylmethane for oligomer-selective dye crystal violet dyes (OSDs). (CV) and Due the to the closely slightly related methylbetter violet signal-to-noise as promising ratio candidates of CV over formethyl oligomer-selective violet, we focused dyes on CV (OSDs). for our Due subsequent to the slightly analysis. better signal-to-noise ratio of CV over methyl violet, we focused on CV for our subsequent analysis. Biomolecules 2019, 9, 539 7 of 15 Biomolecules 2019, 9, x FOR PEER REVIEW 7 of 15

(a) (b) (c) (d)

FigureFigure 4. Fluorescence 4. Fluorescence kinetics kinetics during during sigmoidal sigmoidal and bi biphasicphasic hewL hewL amyloid amyloid assembly assembly for forvarious various fluorescentfluorescent dyes. dyes. Kinetics Kinetics responsesresponses of of (a ()a thioflavin) thioflavin T, ( T,b) the (b) amyloid the amyloid dye X-34 dye (λ X-34ex = 370 (λ exnm,= λ370em = nm, λem 482= 482 nm), nm (c),) acridine (c) acridine orange orange (λex = (495λex nm,= 495 λem nm, = 532λem nm),= 532and nm),(d) acid and fuchsin (d) acid (λex fuchsin = 560 nm, (λ exλem= =560 590 nm, λem nm)= 590 to amyloid nm) to amyloidgrowth of growth hewL at ofthe hewL indicated at the concentrations indicated concentrations (in mg/mL) and (inunder mg fixed/mL) solution and under conditions (pH 2, 52 °C, 450 mM NaCl). Orange (0.3 & 0.6 mg/mL) versus blue traces (2 & 4 mg/mL) fixed solution conditions (pH 2, 52 ◦C, 450 mM NaCl). Orange (0.3 & 0.6 mg/mL) versus blue traces (2 &represent 4 mg/mL) sigmoidal represent versus sigmoidal biphasic versus growth biphasic conditio growthns. Black conditions. traces are dye Black controls traces recorded are dye from controls recordedthe buffer from solution. the buff Forer solution.overall comparison, For overall ThT comparison, traces are displayed ThT traces on a are linear displayed scale, which on a obscures linear scale, its weak biphasic response. Dye concentrations were 15 μM. Sharp initial transients resulted from the which obscures its weak biphasic response. Dye concentrations were 15 µM. Sharp initial transients temperature dependence of dye fluorescence upon heating to 52 °C. resulted from the temperature dependence of dye fluorescence upon heating to 52 ◦C.

3.1.3.1. Crystal Crystal Violet Violet as Oligomer-Selective as Oligomer-Selective Indicator Indicator Dye Dye Figure 5a show the superposition of ThT and CV fluorescence responses during sigmoidal (150 Figure5a show the superposition of ThT and CV fluorescence responses during sigmoidal (150 mM mM NaCl) versus biphasic (450 mM NaCl) amyloid growth. To emphasize the dramatic differences NaCl) versus biphasic (450 mM NaCl) amyloid growth. To emphasize the dramatic differences in in fluorescence responses of the two dyes to sigmoidal versus biphasic amyloid growth, the fractional fluorescence responses of the two dyes to sigmoidal versus biphasic amyloid growth, the fractional fluorescence increases F/F0 for either dye are displayed on a semi-logarithmic scale. There are several fluorescenceindications increases that theF distinct/F0 for eitherdye kinetics dye are record displayeded with on CV a semi-logarithmic versus ThT during scale. sigmoidal There areversus several indicationsbiphasic that growth the distinctare due dyeto the kinetics differential recorded sensitivity with CVof CV versus to gO/CF ThT duringversus RF sigmoidal formation. versus Based biphasic on growththe near-identical are due to the 400-fold differential increase sensitivity in ThT amplitud of CVes to at gO the/CF end versus of the RF96 h formation. incubation period, Based the on the near-identicalamount of 400-foldamyloid fibrils increase present in ThT in the amplitudes 150 versus at 450 the mM end salt of thesolutions 96 h incubation are comparable period, (Figure the amount5a). of amyloidIn contrast, fibrils CV fluorescence present in the in the 150 biphasic versus regi 450me mM predominately salt solutions responded are comparable to the initial (Figure oligomer-5a). In contrast,related CV upswing fluorescence and inwith the biphasica roughly regime 10-times predominately larger response responded than to to fibril the initial formation oligomer-related during upswingsigmoidal and fibril with growth. a roughly This 10-times difference larger in CV response response than persisted to fibril irrespective formation of whether during sigmoidalThT and CV fibril growth.were Thisadded di tofference the same in sample CV response wells or persisted were measured irrespective in separate of whether sample ThTwells, and thereby, CV were excluding added to the contributions from the competitive binding of CV versus ThT to the fibrils. the same sample wells or were measured in separate sample wells, thereby, excluding the contributions from the competitive binding of CV versus ThT to the fibrils.

The muted CV response to RF formation could potentially arise if CV acts as a select inhibitor of RF formation. The strong ThT response, recorded in the presence of CV under the same condition (see Figure5a) contradicts that interpretation. To further exclude that CV inhibits RF growth or alters ThT responses, we monitored sigmoidal RF growth for identical hewL/solution conditions, at fixed ThT concentration, but for a series of increasing CV concentrations. As shown in Figure5b, CV had no discernible effect on the RF kinetics recorded with ThT, up to twice the 5 µM CV concentration used in our typical experiments. At 30 µM of CV, ThT amplitudes were slightly reduced, but with the kinetics of ThT recordings still unchanged. Hence, for the dye concentrations used in our experiments, there was no indication that CV inhibited RF formation. As has been reported for Congo Red and ThT binding to Aβ42 [53], the reduced ThT response observed at 30 µM of CV most likely reflected the competitive binding of ThT and CV to fibrils, instead of the CV-inhibiting RF formation. Another notable feature of the CV signal during the biphasic growth is its persistence, well into the late-stage RF nucleation and growth period as seen with ThT. We have previously shown that gO/CFs, formed during the initial phase of biphasic kinetics, persist long into the RF nucleation and growth-dominated second phase, including the late-stage plateau in the ThT responses [41]. This reflects the slow rate of gO/CF depolymerization and gradual incorporation into RFs. We reconfirm this prolonged persistence of gO/CFs into the RF growth phase, using a “staggered” incubation protocol—the same biphasic growth solution was added at progressively later times to a temperature-controlled 96-well plate and the accumulated aggregates were collected at a single Biomolecules 2019, 9, 539 8 of 15 time-point for imaging. Figure6a shows the associated series of staggered ThT and CV time traces, Biomolecules 2019, 9, x FOR PEER REVIEW 9 of 15 together with the correlated AFM images of aggregate morphologies representing different time-points in the assemblySince CV reaction. and ThT Consistentrespond to both with gO/CFs our previous and RFs, observations,we evaluated whether the initial both phase dyes provide of biphasic kineticsidentical was dominated assembly kinetics by gO/ CFbut formation,with different while sensitivities. the second For phase this, indicatedwe determined nucleation the temporal and growth of RFs,correlation with residual between populations the CV and of the gO ThT/CFs amplitudes persisting under into the either late-stages the sigmoidal of our or kinetic biphasic traces. growth Hence, the initialconditions. rise of As CV shown fluorescence in Figure during 5c, the the two biphasic dye signals growth were correlated perfectly withcorrelated the emergence in the sigmoidal of gO /CFs. Biomolecules 2019, 9, x FOR PEER REVIEW 8 of 15 The persistenceregime, but ofwith CV CV fluorescence showing a much throughout weaker theRF response. nucleation During and growththe biphasic of RFs growth, detected in contrast, by the ThT was supporteddye responses by showed the prolonged a 1:1 correlation presence in ofdye gO respon/CFsse in during the AFM the imagesinitial gO/CF during growth that phase, period. while the two dye signals nearly completely uncoupled during the subsequent RF nucleation and growth phase. To further confirm this preferential selectivity of CV for gO/CFs over RFs, we repeated the experiments by fixing solution conditions and varying the hewL concentrations, instead. Figure 7a,b compared the ThT and CV traces obtained from the same wells. After accounting for their relative difference in sensitivity and a slight shift in initial response time, the fractional CV and ThT increases were perfectly correlated during the sigmoidal RF growth. This perfect correlation in the sigmoidal regime indicated that both dyes detected the same aggregate populations over the entire time course of nucleated RF polymerization. Plotting the fractional ThT versus CV amplitudes for the sigmoidal growth in Figure 7e, further emphasizes that the two dye signals were perfectly linearly correlated over the nearly 7-day incubation period, albeit with very different fluorescence enhancements. The (c) corresponding correlation(a) coefficient of ΓRF = 0.013(b) indicates that the CV response under these FigureconditionsFigure 5. Crystal was 5. Crystal about violet violet75 times as as the the weaker oligomer-selective oligomer-selective than the ThT dye dye response (OSD). (OSD). (a )to Fractional (equivalenta) Fractional change concentrations change of 15 μM of ThT 15 of (openµ RFs.M ThT In the biphasic growth regime (blue traces in Figure 7a,b), ThT and CV displayed equivalent (opensymbols) symbols) versus versus 5 μ 5Mµ crystalM crystal violet violet (filled (filled symbols) symbols) fluorescence fluorescence during sigmoidal during versus sigmoidal biphasic versus kineticsamyloid during assembly the oligomer-dominated (350 μM hewL, pH 2,initial 52 °C) phase, in the presence with an ofoverall either 15020-fold or 400 fractional mM NaCl). fluorescence (b) ThT biphasic amyloid assembly (350 µM hewL, pH 2, 52 ◦C) in the presence of either 150 or 400 mM NaCl). enhancement(15 μM) andkinetics a linear of hewL correlation undergoing coefficient sigmoidal of ΓfibrilgO/CF growth= 0.81 ±in 0.01.the presenceThe dramatically of increasing lower (b) ThT (15 µM) kinetics of hewL undergoing sigmoidal fibril growth in the presence of increasing sensitivityconcentrations of CV versus of CV. ThT (c )to Correlation subsequent of RFfractional nucleation CV versus and grow ThT thaugmentation resulted in duringthe near-complete biphasic concentrations of CV. (c) Correlation of fractional CV versus ThT augmentation during biphasic versus uncouplingversus ofsigmoidal their respective growth. fluorescenceDuring the oligomer-dom responses ininated the second, phase RF-dominatedof biphasic kinetics, phase. fractional However, sigmoidal growth. During the oligomer-dominated phase of biphasic kinetics, fractional changes of CV the ThTchanges picked of up CV the and secondary, ThT fluorescence fibril-dominated are in a lock phstepase and (Figure are of comparable7a), the low magnitude. sensitivity In of contrast, CV to RFs andand ThTtheCV concomitant fluorescencebecomes essentially decay are in unresponsiveof a lockgO/CFs step (see and to the Figure are second, of comparable6 above) fibril-dominated induced magnitude. aphase. subtle InDuring decay contrast, sigmoidal of CV CV upon becomesfibril the essentiallyemergencegrowth, unresponsiveof CVRFs barely (Figure increases to 7b). the The second, 1.5-fold, corresponding fibril-dominatedcompared toamplitude-matched the nearly phase. 100-fold During fluorescencesuperposition sigmoidal increase fibril of CV growth, of and ThT. ThT CV barelytraces Upon increasesis shown aligning 1.5-fold,in Figure the (noise-limited) compared 7d. The uncoupling to theCV nearlytraces of with 100-foldThT the and upswing fluorescence CV responses in ThT, increase intheir the responses oflate ThT. phaseUpon are of strictly biphasic aligning thegrowth (noise-limited)linearly is reflected correlated CV similarly tracesin time. with in the the sharp upswing break in ThT,in the their correlation responses of are the strictly fractional linearly fluorescence correlated inincreases time. for ThT versus CV between the initial and secondary phases of biphasic growth (Figure 7f). The muted CV response to RF formation could potentially arise if CV acts as a select inhibitor of RF formation. The strong ThT response, recorded in the presence of CV under the same condition (see Figure 5a) contradicts that interpretation. To further exclude that CV inhibits RF growth or alters ThT responses, we monitored sigmoidal RF growth for identical hewL/solution conditions, at fixed ThT concentration, but for a series of increasing CV concentrations. As shown in Figure 5b, CV had no discernible effect on the RF kinetics recorded with ThT, up to twice the 5 μM CV concentration used in our typical experiments. At 30 μM of CV, ThT amplitudes were slightly reduced, but with the kinetics of ThT recordings still unchanged. Hence, 5for h the dye concentrations28 h used in our experiments, there was no indication that CV inhibited RF formation. As has been reported for Congo Red and ThT binding to Aβ42 [53], the reduced ThT response observed at 30 μM of CV most likely reflected the competitive binding of ThT and CV to fibrils, instead of the CV-inhibiting RF formation.

Another notable feature of the CV signal during the biphasic growth is its persistence, well into the late-stage RF nucleation and growth period as seen with ThT. We have previously shown that gO/CFs, formed during the initial phase of biphasic kinetics, persist long into the RF nucleation and 52 h 97 h growth-dominated second phase, including the late-stage plateau in the ThT responses [41]. This (a) (b) reflects the slow rate of gO/CF depolymerization and gradual incorporation into RFs. We reconfirm FigurethisFigure prolonged 6. Evolution 6. Evolution persistence of amyloid of amyloid of aggregate gO/CFs aggregate morphologyinto themorphology RF duringgrowth during biphasic phase, biphasic kineticsusing kineticsa( a“staggered”) Staggered (a) Staggered incubationincubation protocolincubation—the of same 350 μ Mbiphasic hewL undergoing growth biphasicsolution amyloid was addedgrowth atat pH progressively 2, 52 °C, and 450later mM timesNaCl, to a of 350 µM hewL undergoing biphasic amyloid growth at pH 2, 52 ◦C, and 450 mM NaCl, recorded temperature-controlledrecorded simultaneously 96-well with 15plate μM and ThT the () accumula and 5 μMted CV aggregates(). Fresh solutions were collected were added at a singleto the time- simultaneously with 15 µM ThT () and 5 µM CV (). Fresh solutions were added to the 96-well plates point96-well for imaging.plates at theFigure moments 6a shows indicated the by associated the arrows. series Corresponding of staggered total incubationThT and periodsCV time are traces, at the moments indicated by the arrows. Corresponding total incubation periods are shown next to togethershown with next tothe each correlated arrow. (b )AFM AFM imagesimages of of aliquots aggregate imaged morphologies from wells incubated representing for the differentindicated time- each arrow. (b) AFM images of aliquots imaged from wells incubated for the indicated time periods. pointstime in periods. the assembly While the reaction.initial phase Consistent of biphasic with growth our indicates previous the observations,presence of gOs the and initialincreasing phase of While the initial phase of biphasic growth indicates the presence of gOs and increasing numbers of CFs, biphasicnumbers kinetics of CFs, was the dominated late phase showsby gO/CF the simultanformatioeousn, while presence the ofsecond RFs and phase CFs, indicated often in direct nucleation the late phase shows the simultaneous presence of RFs and CFs, often in direct contact with each other. and growth of RFs, with residual populations of gO/CFs persisting into the late-stages of our kinetic µ Thetraces. false Hence, color scale the indicatesinitial rise aggregate of CV fluorescence heights. All imagesduring arethe 3biphasicm on a growth side. correlated with the emergence of gO/CFs. The persistence of CV fluorescence throughout the nucleation and growth of RFs detected by the ThT was supported by the prolonged presence of gO/CFs in the AFM images during that period.

3.2. CV and ThT Report Identical Amyloid Kinetics but with Distinctly Different Sensitivities Biomolecules 2019, 9, 539 9 of 15 Biomolecules 2019, 9, x FOR PEER REVIEW 10 of 15

contact with each other. The false color scale indicates aggregate heights. All images are 3 μm on a 3.2. CV and ThT Report Identical Amyloid Kinetics but with Distinctly Different Sensitivities side. Since CV and ThT respond to both gO/CFs and RFs, we evaluated whether both dyes provide identical3.3. Separating assembly Amyloid kinetics Assembly but with into di ffitserent gO/CF sensitivities. and RF Components For this, we determined the temporal correlationThe tight between temporal the CV correlation and the ThTof CV amplitudes and ThT re undersponses either to RF the formation, sigmoidal combined or biphasic with growththeir conditions.distinctly Asdifferent shown sensitivities in Figure5 c,to the gO/CFs two dyeover signals RFs, suggests were perfectly that simultaneous correlated in CV the and sigmoidal ThT regime,recordings but with could CV permit showing decomposition a much weaker of RFbiphasic response. kinetics During into the separate biphasic gO/CF growth, and in RFs contrast, growth dye responseskinetics. showedThis is important a 1:1 correlation since the in temporal dye response evolution during of gO/CFs the initial and gO RFs/CF typically growth overlap phase, whilein time the twoand, dye as signals we have nearly previously completely suggested, uncoupled there duringare indications the subsequent that gO/CFs RF nucleation directly interact and growth with and phase. Tomodulate further confirm the kinetics this preferential of subseque selectivitynt RF nucleation of CV for and gO /growthCFs over [41]. RFs, To we test repeated this, we the amplitude- experiments bymatched fixing solution the initial conditions oligomer-dominated and varying phase the hewLof the CV concentrations, trace to its corresponding instead. Figure ThT7 tracea,b compared (Figure the7d), ThT using and CVtheir traces correlation obtained of 0.81 from (Figure the same 7f). wells.As can After be seen, accounting the two formatched their relative signals dicoincidedfference in sensitivityperfectly and during a slight the initial shift in portion initial of response biphasic time, growth the fractionaland then rapidly CV and diverged. ThT increases The orange were perfectlycurve correlatedin Figure during 7d is the the difference sigmoidal trace RF growth. resulting This from perfect subtracting correlation the matched in the sigmoidal CV from the regime ThT indicatedsignal. thatEven both though dyes this detected approach the sameignores aggregate the (weak) populations contributions over of the RF entire formation time course to the ofoligomeric nucleated CV signal, the difference curve in Figure 7d readily recovers the characteristic sigmoidal kinetics of RF polymerization. Plotting the fractional ThT versus CV amplitudes for the sigmoidal growth in RF formation—a lag period flat down to the noise level of the fluorescence signal and a rapid Figure7e, further emphasizes that the two dye signals were perfectly linearly correlated over the nearly autocatalytic upswing nearly reaching the plateau-level of the ThT signal. The blue CV and orange 7-day incubation period, albeit with very different fluorescence enhancements. The corresponding (ThT–CV) difference traces, therefore, represent a near-perfect decomposition of the hewL amyloid correlation coefficient of Γ = 0.013 indicates that the CV response under these conditions was about kinetics into its separateRF gO/CF and RF growth components—without any a priori assumption 75concerning times weaker the thanunderl theying ThT gO/CF response kinetics. to equivalent concentrations of RFs.

(a) (b) (c)

(d) (e) (f)

FigureFigure 7. 7.Correlation Correlation of of CVCV andand ThT kinetics. (a (,ad,d) )HewL HewL amyloid amyloid formation formation (pH (pH 2, 450 2, 450mM mMNaCl, NaCl, T = 52 °C) at concentrations below (orange) and above (blue) the COC, simultaneously monitored with T = 52 ◦C) at concentrations below (orange) and above (blue) the COC, simultaneously monitored with (a) ThT (15 μM) and (d) CV (5 μM). The traces are the average of the three recordings from separate (a) ThT (15 µM) and (d) CV (5 µM). The traces are the average of the three recordings from separate wells. (b,e) Superposition of the CV (solid lines) and ThT (dashed lines) responses, after matching the wells. (b,e) Superposition of the CV (solid lines) and ThT (dashed lines) responses, after matching the CV data using the Γ-factor determined in (c) and (f), respectively. Subtracting the matched CV from CV data using the Γ-factor determined in (c) and (f), respectively. Subtracting the matched CV from the ThT trace yields the orange sigmoidal trace in (e). (c,f) Correlation of the fractional changes of CV the ThT trace yields the orange sigmoidal trace in (e). (c,f) Correlation of the fractional changes of versus ThT responses during (c) sigmoidal versus (f) biphasic growth kinetics. The Γ-coefficient CV versus ThT responses during (c) sigmoidal versus (f) biphasic growth kinetics. The Γ-coefficient indicates the ratio of the CV versus ThT response amplitude in the linear regime of each plot. indicates the ratio of the CV versus ThT response amplitude in the linear regime of each plot. 3.4. Biphasic Kinetics of Alzheimer Peptides Aβ40 and Aβ42 upon Onset of gO/CF Formation In the biphasic growth regime (blue traces in Figure7a,b), ThT and CV displayed equivalent kineticsThere during are the two oligomer-dominated intrinsic shortcomings initial of using phase, hewL with to anscreen overall for oligomer-selective 20-fold fractional dyes. fluorescence First, enhancementwhile hewL is and readily a linear available correlation and its coeassemblyfficient kineti of Γcs are highly= 0.81 reproducible,0.01. The it dramatically is not a clinically lower gO/CF ± sensitivity of CV versus ThT to subsequent RF nucleation and growth resulted in the near-complete Biomolecules 2019, 9, 539 10 of 15 uncoupling of their respective fluorescence responses in the second, RF-dominated phase. However, the ThT picked up the secondary, fibril-dominated phase (Figure7a), the low sensitivity of CV to RFs and the concomitant decay of gO/CFs (see Figure6 above) induced a subtle decay of CV upon the emergence of RFs (Figure7b). The corresponding amplitude-matched superposition of CV and ThT traces is shown in Figure7d. The uncoupling of ThT and CV responses in the late phase of biphasic growth is reflected similarly in the sharp break in the correlation of the fractional fluorescence increases for ThT versus CV between the initial and secondary phases of biphasic growth (Figure7f).

3.3. Separating Amyloid Assembly into its gO/CF and RF Components The tight temporal correlation of CV and ThT responses to RF formation, combined with their distinctly different sensitivities to gO/CFs over RFs, suggests that simultaneous CV and ThT recordings could permit decomposition of biphasic kinetics into separate gO/CF and RFs growth kinetics. This is important since the temporal evolution of gO/CFs and RFs typically overlap in time and, as we have previously suggested, there are indications that gO/CFs directly interact with and modulate the kinetics of subsequent RF nucleation and growth [41]. To test this, we amplitude-matched the initial oligomer-dominated phase of the CV trace to its corresponding ThT trace (Figure7d), using their correlation of 0.81 (Figure7f). As can be seen, the two matched signals coincided perfectly during the initial portion of biphasic growth and then rapidly diverged. The orange curve in Figure7d is the difference trace resulting from subtracting the matched CV from the ThT signal. Even though this approach ignores the (weak) contributions of the RF formation to the oligomeric CV signal, the difference curve in Figure7d readily recovers the characteristic sigmoidal kinetics of RF formation—a lag period flat down to the noise level of the fluorescence signal and a rapid autocatalytic upswing nearly reaching the plateau-level of the ThT signal. The blue CV and orange (ThT–CV) difference traces, therefore, represent a near-perfect decomposition of the hewL amyloid kinetics into its separate gO/CF and RF growth components—without any a priori assumption concerning the underlying gO/CF kinetics.

3.4. Biphasic Kinetics of Alzheimer Peptides Aβ40 and Aβ42 upon Onset of gO/CF Formation There are two intrinsic shortcomings of using hewL to screen for oligomer-selective dyes. First, while hewL is readily available and its assembly kinetics are highly reproducible, it is not a clinically relevant amyloid protein. This shortcoming is mitigated by the observation that the common amyloid stains ThT and Congo Red, as well as some of the newly identified amyloid dyes, often detect amyloids formed by various proteins [38]. The second shortcoming are the non-physiological solution conditions used to observe sigmoidal versus biphasic growth with hewL. As mentioned above, the oligomers formed under those conditions match the features of the toxic oligomers. However, it is well-known that dye responses can vary significantly with solution conditions, such as temperature, pH and ionic strength. However, similar to β2-microglobulin, hewL forms gO/CFs or precipitates near neutral pH—without any discernible RF populations or kinetics to compare to [54]. Hence, we could not extend our kinetic assay to a neutral pH using hewL. We, therefore, explored whether the above kinetic transition could be observed with the clinically relevant Alzheimer Disease peptides Aβ40 and Aβ42, under physiological growth conditions. As shown in Figure8, both A β40 and Aβ42 did display a transition from sigmoidal to biphasic kinetics upon increasing peptide concentration. Using TEM we confirmed that Aβ40 solutions in the sigmoidal regime only yielded fibrillar aggregates (Figure8d). Biomolecules 2019, 9, x FOR PEER REVIEW 11 of 15 relevant amyloid protein. This shortcoming is mitigated by the observation that the common amyloid stains ThT and Congo Red, as well as some of the newly identified amyloid dyes, often detect amyloids formed by various proteins [38]. The second shortcoming are the non-physiological solution conditions used to observe sigmoidal versus biphasic growth with hewL. As mentioned above, the oligomers formed under those conditions match the features of the toxic oligomers. However, it is well-known that dye responses can vary significantly with solution conditions, such as temperature, pH and ionic strength. However, similar to β2-microglobulin, hewL forms gO/CFs or precipitates near neutral pH—without any discernible RF populations or kinetics to compare to [54]. Hence, we could not extend our kinetic assay to a neutral pH using hewL. We, therefore, explored whether the above kinetic transition could be observed with the clinically relevant Alzheimer Disease peptides Aβ40 and Aβ42, under physiological growth conditions. As shown in Figure 8, both Aβ40 and Aβ42 did display a transition from sigmoidal to biphasic kinetics upon increasing peptide concentration. Using TEM we confirmed that Aβ40 solutions in the sigmoidal regime only yielded fibrillar aggregates (Figure 8d). Upon the transition to biphasic growth, initial aggregate populations were dominated by highly curvilinear fibrils (CFs) which, even after 6 days, was only partially superseded by RFs (Figure 8e,f). In addition, the initial, oligomeric phase of Aβ40 amyloid growth was further enhanced upon using physiological NaCl concentrations (150 mM). These observations matched the reports of micelle-like oligomer formation in these systems [43,44] and the kinetic measurements in either system [45,55,56]. However, while the gO/CF and RF growth phases of Aβ40 were well-separated by a clear plateau phase (Figure 8a,c), those of Aβ42 showed a strong overlap (Figure 8b). In order to separate the dye responses into their gO/CF versus RF components, a clear temporal separation of the two growth phases is highly desirable. These findings with Aβ40 and Aβ42 are consistent with the switch from oligomer-free sigmoidal to oligomer-dominated biphasic growth that we observed for dimAβ and hewL. Hence, the screening approach presented here can be extended to screen for novel OSDs with Biomolecules 2019, 9, 539 11 of 15 Aβ, under physiological solution conditions.

Figure 8. Transition from sigmoidal to biphasic kineticskinetics and associated gOgO/CF/CF formation for Aβ40 and A β42. ( (aa)) Transition Transition in in A Aββ4040 growth growth kinetics kinetics from from pure pure sigmoid sigmoidalal (orange) (orange) to to biphasic (blue) kinetics (pH 7.4, no salt). ( b) Same as (a) but forfor AAβ42. Semilog plot emphasizes weak ThT response during gOgO/CF/CF phase.phase. ((cc)) ThT ThT fractional fractional change change during during A βA40β40 growth growth in physiologicalin physiological saline. saline. Notice Notice the thesigniﬁcant significant increase increase in gO in/ CFgO/CF amplitude amplitude relative relative to panel to panel (a). ( d(a–).f) ( TEMd–f) TEM images images of samples of samples of (a)—A of (aβ)—40 ARFsβ40 following RFs following sigmoidal sigmoidal growth growth at 50 µ atM( 50d )μ versusM (d) versus biphasic biphasic growth growth at 150 µ atM, 150 with μM, gO with/CFs gO/CFs formed withinformed1.5 within days, 1.5 and days, (e) mixturesand (e) mixtures of gO/CF of and gO/CF RFs and after RFs 6 days after ( f6). days (f).

4. DiscussionUpon the transition to biphasic growth, initial aggregate populations were dominated by highly curvilinear ﬁbrils (CFs) which, even after 6 days, was only partially superseded by RFs (Figure8e,f). In addition, the initial, oligomeric phase of Aβ40 amyloid growth was further enhanced upon using physiological NaCl concentrations (150 mM). These observations matched the reports of micelle-like oligomer formation in these systems [43,44] and the kinetic measurements in either system [45,55,56]. However, while the gO/CF and RF growth phases of Aβ40 were well-separated by a clear plateau phase (Figure8a,c), those of A β42 showed a strong overlap (Figure8b). In order to separate the dye responses into their gO/CF versus RF components, a clear temporal separation of the two growth phases is highly desirable. These ﬁndings with Aβ40 and Aβ42 are consistent with the switch from oligomer-free sigmoidal to oligomer-dominated biphasic growth that we observed for dimAβ and hewL. Hence, the screening approach presented here can be extended to screen for novel OSDs with Aβ, under physiological solution conditions.

4. Discussion One of the persistent challenges in amyloid research is to unravel the enigmatic role of oligomeric species as either competitors or on-pathway contributors to amyloid fibril assembly, as well as their contributions to the etiology of amyloid diseases in vivo. As pointed out above, part of the challenge is the dependence on antibodies as the exclusive means for detecting and tracking oligomer populations, particularly in in vivo studies. The data presented here suggest that we can identify and validate novel, small oligomer-selective dyes by determining their fluorescence responses to sigmoidal versus biphasic growth. This kinetic approach to dye screening circumvents the need to isolate and stabilize transient and metastable oligomers, in order to screen dyes for their oligomer selectivity. The assay is also robust since both the sigmoidal and biphasic kinetics are highly reproducible for a given choice of solution conditions. Using this approach, we identified the triarylmethane dye crystal violet (CV), as highly selective to lysozyme oligomers over fibrils. We further confirmed that, in the fibril-associated sigmoidal assembly regime, CV yielded kinetics identical to those of ThT—albeit with a significantly Biomolecules 2019, 9, 539 12 of 15 lower sensitivity. In contrast, the sensitivity of either dye to the oligomer-dominated initial phase of biphasic growth were nearly identical. While a higher oligomer-sensitivity would be desirable, CV clearly displaced strong selectivity for gO/CFs over RFs in its fluorescence response. This gO/CF selectivity already allowed us to decompose simultaneous CV and ThT recordings of biphasic kinetics into their separate gO/CF and RF components. This should boost efforts in studying the kinetics of oligomer formation versus fibril growth and in the determination of when and how these two processes interact with each other. In addition, this assay could serve as a high throughput screen for monitoring the effects of putative drug candidates on amyloid oligomers versus fibril formation. Perhaps most importantly, though, identifying new small molecules that bind selectivity to oligomers could serve as a starting point in the development of positron-emission tomography (PET) probes, for monitoring the emergence and spread of oligomeric over fibrillar species in neurodegenerative diseases, in vivo.

5. Conclusions Using the readily available amyloid model hewL, we provided a proof of principle for using sigmoidal versus biphasic kinetics as a screening assay for oligomer-selective dyes. While selective under multiple conditions, we did find that the relative selectivity of CV versus ThT for hewL gO/CFs and RFs did vary with the ionic strength of the growth solution. It is known that the binding of dyes or their fluorescence responses vary with the solution pH and temperature [57,58]. Therefore, for a given amyloid protein, it is important to establish selectivity under physiological growth conditions. To address this concern, we confirmed that the same kinetic transition from sigmoidal to biphasic kinetics is present in Aβ40 and Aβ42 at physiological pH and ionic strength. Hence, we are confident that the screening and validation approach presented here could be extended at least to the Alzheimer’s disease relevant Aβ peptides and are also relevant to other disease-associated amyloid proteins.

Author Contributions: Conceptualization, M.M. and J.B.; methodology, M.M, J.B., and D.S.A.; formal analysis, J.B., D.S.A, and C.N.; investigation, J.B., C.N., G.B., N.L., and S.M.; writing—original draft preparation, M.M., J.B., and C.N.; writing—review and editing, M.M. and J.B.; supervision, M.M.; project administration, M.M.; funding acquisition, M.M. Funding: This research was funded by the National Institutes of Health grant 2R15GM097723-02. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Knowles, T.P.J.; Vendruscolo, M.; Dobson, C.M. The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 2014, 15, 384–396. [CrossRef][PubMed] 2. Blancas-Mejia, L.M.; Ramirez-Alvarado, M. Systemic Amyloidoses. Annu. Rev. Biochem. 2013, 82, 745–774. [CrossRef][PubMed] 3. Invernizzi, G.; Papaleo, E.; Sabate, R.; Ventura, S. Protein aggregation: Mechanisms and functional consequences. Int. J. Biochem. Cell Biol. 2012, 44, 1541–1554. [CrossRef][PubMed] 4. Aguzzi, A.; O’Connor, T. Protein aggregation diseases: Pathogenicity and therapeutic perspectives. Nat Rev Drug Discov. 2010, 9, 237–248. [CrossRef][PubMed] 5. Ross, C.A.; Poirier, M.A. Protein aggregation and neurodegenerative disease. Nat. Med. 2004, 10, S10–S17. [CrossRef] 6. Dobson, C.M. Protein misfolding, evolution and disease. Trends Biochem. Sci. 1999, 24, 329–332. [CrossRef] 7. Chiti, F.; Dobson, C.M. Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Rev. Biochem. 2006, 75, 333–366. [CrossRef] 8. Hardy, J.; Selkoe, D.J. The Amyloid Hypothesis of Alzheimer’s Disease: Progress and Problems on the Road to Therapeutics. Science 2002, 297, 353–356. [CrossRef] 9. Selkoe, D.J. Alzheimer’s Disease: Genes, Proteins, and Therapy. Physiol. Rev. 2001, 81, 741–766. [CrossRef] 10. Cornwell, G.G., III; Johnson, K.H.; Westermark, P. The age related amyloids: A growing family of unique biochemical substances. J. Clin. Pathol. 1995, 48, 984–989. [CrossRef] Biomolecules 2019, 9, 539 13 of 15

11. Hull, R.L.; Kahn, S.E.; Westermark, G.T.; Westermark, P. Islet Amyloid: A Critical Entity in the Pathogenesis of Type 2 Diabetes. J. Clin. Endocrinol. Metab. 2004, 89, 3629–3643. [CrossRef] 12. Haass, C.; Selkoe, D.J. Soluble protein oligomers in neurodegeneration: Lessons from the Alzheimer’s amyloid beta-peptide. Nat. Rev. Mol. Cell Biol. 2007, 8, 101–112. [CrossRef] 13. Lesné, S.; Koh, M.T.; Kotilinek, L.; Kayed, R.; Glabe, C.G.; Yang, A.; Gallagher, M.; Ashe, K.H. A specific amyloid-[beta] protein assembly in the brain impairs memory. Nature 2006, 440, 352–357. [CrossRef] 14. Tomic, J.L.; Pensalfini, A.; Head, E.; Glabe, C.G. Soluble fibrillar oligomer levels are elevated in Alzheimer’s disease brain and correlate with cognitive dysfunction. Neurobiol. Dis. 2009, 35, 352–358. [CrossRef] [PubMed] 15. Dahlgren, K.N.; Manelli, A.M.; Stine, W.B.; Baker, J.; Lorinda, K.; Krafft, G.A.; LaDu, M.J. Oligomeric and Fibrillar Species of Amyloid-β Peptides Differentially Affect. Neuronal Viability. J. Biol. Chem. 2002, 277, 36046–36053. [CrossRef] 16. Cleary, J.P.; Walsh, D.M.; Hofmeister, J.J.; Shankar, G.M.; Kuskowski, M.A.; Selkoe, D.J.; Ashe, K.H. Natural oligomers of the amyloid-β protein specifically disrupt cognitive function. Nat. Neurosci. 2005, 8, 79–84. [CrossRef][PubMed] 17. Pigino, G.; Morfini, G.; Atagi, Y.; Deshpande, A.; Yu, C.; Jungbauer, L.; LaDu, M.; Busciglio, J.; Brady, S. Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta. Proc. Natl. Acad. Sci. USA 2009, 106, 5907–5912. [CrossRef][PubMed] 18. Kayed, R.; Lasagna-Reeves, C.A. Molecular Mechanisms of Amyloid Oligomers Toxicity. J. Alzheimers Dis. 2013, 33, S67–S78. [CrossRef] 19. Serrano-Pozo, A.; Frosch, M.P.; Masliah, E.; Hyman, B.T. Neuropathological Alterations in Alzheimer Disease. Cold Spring Harb. Perspect. Med. 2011, 1, a006189. [CrossRef][PubMed] 20. Hefti, F.; Goure, W.F.; Jerecic, J.; Iverson, K.S.; Walicke, P.A.; Krafft, G.A. The case for soluble Aβ oligomers as a drug target in Alzheimer’s disease. Trends Pharmacol. Sci. 2013, 34, 261–266. [CrossRef][PubMed] 21. Harper, J.D.; Wong, S.S.; Lieber, C.M.; Lansbury, P.T. Observation of metastable Aβ amyloid protofibrils by atomic force microscopy. Chem. Biol. 1997, 4, 119–125. [CrossRef] 22. Stine, W.B., Jr.; Dahlgren, K.N.; Krafft, G.A.; LaDu, M.J. In Vitro Characterization of Conditions for Amyloid-beta Peptide Oligomerization and Fibrillogenesis. J. Biol. Chem. 2003, 278, 11612–11622. [CrossRef] [PubMed] 23. Hill, S.E.; Robinson, J.; Matthews, G.; Muschol, M. Amyloid Protofibrils of Lysozyme Nucleate and Grow Via Oligomer Fusion. Biophys. J. 2009, 96, 3781–3790. [CrossRef][PubMed] 24. Bitan, G. Structural Study of Metastable Amyloidogenic Protein Oligomers by Photo-Induced Cross-Linking of Unmodified Proteins. In Methods in Enzymology; Academic Press: Cambridge, MA, USA, 2006; pp. 217–236. 25. Young, L.M.; Cao, P.; Raleigh, D.P.; Ashcroft, A.E.; Radford, S.E. Ion. Mobility Spectrometry–Mass Spectrometry Defines the Oligomeric Intermediates in Amylin Amyloid Formation and the Mode of Action of Inhibitors. J. Am. Chem. Soc. 2014, 136, 660–670. [CrossRef][PubMed] 26. Cerf, E.; Sarroukh, R.; Tamamizu-Kato, S.; Breydo, L.; Derclaye, S.; Dufrêne, Y.F.; Narayanaswami, V.; Goormaghtigh, E.; Ruysschaert, J.; Raussens, V. Antiparallel β-sheet: A signature structure of the oligomeric amyloid β-peptide. Biochem. J. 2009, 421, 415–423. [CrossRef] 27. Foley, J.; Hill, S.E.; Miti, T.; Mulaj, M.; Ciesla, M.; Robeel, R.; Persichilli, C.; Raynes, R.; Westerheide, S.; Muschol, M. Structural Fingerprints and their Evolution during Oligomeric vs. Oligomer-free Amyloid Fibril Growth. J. Chem. Phys. 2013, 139, 121901. [CrossRef][PubMed] 28. Kayed, R.; Head, E.; Sarsoza, F.; Saing, T.; Cotman, C.; Necula, M.; Margol, L.; Wu, J.; Breydo, L.; Thompson, J.; et al. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol. Neurodegener. 2007, 2, 18. [CrossRef] 29. Kayed, R.; Glabe, C.G. Conformation-Dependent Anti-Amyloid Oligomer Antibodies. In Methods in Enzymology; Academic Press: Cambridge, MA, USA, 2006; pp. 326–344. 30. LeVine, H. Thioflavine T interaction with amyloid β-sheet structures. Amyloid 1995, 2, 1–6. [CrossRef] 31. Groenning, M. Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils—Current status. J. Chem. Biol. 2010, 3, 1–18. [CrossRef] 32. Meisl, G.; Kirkegaard, J.B.; Arosio, P.; Michaels, T.C.T.; Vendruscolo, M.; Dobson, C.M.; Linse, S.; Knowles, T.P.J. Molecular mechanisms of protein aggregation from global fitting of kinetic models. Nat. Protoc. 2016, 11, 252–272. [CrossRef] Biomolecules 2019, 9, 539 14 of 15

33. Knowles, T.P.J.; White, D.A.; Abate, A.R.; Agresti, J.J.; Cohen, S.I.A.; Sperling, R.A.; de Genst, E.J.; Dobson, C.M.; Weitz, D.A. Observation of spatial propagation of amyloid assembly from single nuclei. Proc. Natl. Acad. Sci. USA 2011, 108, 14746–14751. [CrossRef] 34. Yamamoto, T.; Hirano, A. A Comparative Study of Modified Bielschowsky, Bodian and Thioflavin S Stains on Alzheimer’s Neurofibrillary Tangles. Neuropathol. Appl. Neurobiol. 1986, 12, 3–9. [CrossRef][PubMed] 35. Urbanc, B.; Cruz, L.; Le, R.; Sanders, J.; Ashe, K.H.; Duff, K.; Stanley, H.E.; Irizarry, M.C.; Hyman, B.T. Neurotoxic effects of thioflavin S-positive amyloid deposits in transgenic mice and Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 2002, 99, 13990–13995. [CrossRef][PubMed] 36. Klunk, W.E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.; Holt, D.P.; Bergström, M.; Savitcheva, I.; Huang, G.-F.; Estrada, S.; et al. Imaging brain amyloid in Alzheimer’s disease with Pittsburgh Compound-B. Ann. Neurol. 2004, 55, 306–319. [CrossRef][PubMed] 37. Wu, C.; Wei, J.; Gao, K.; Wang, Y. Dibenzothiazoles as novel amyloid-imaging agents. Bioorganic Med. Chem. 2007, 15, 2789–2796. [CrossRef] 38. Hammarström, P.; Simon, R.; Nyström, S.; Konradsson, P.; Åslund, A.; Nilsson, K.P.R. A Fluorescent Pentameric Thiophene Derivative Detects in Vitro-Formed Prefibrillar Protein Aggregates. Biochemistry 2010, 49, 6838–6845. [CrossRef] 39. Younan, N.D.; Viles, J.H. A Comparison of Three Fluorophores for the Detection of Amyloid Fibers and Prefibrillar Oligomeric Assemblies. ThT (Thioflavin T); ANS (1-Anilinonaphthalene-8-sulfonic Acid); and

bisANS (4,40-Dianilino-1,10-binaphthyl-5,50-disulfonic Acid). Biochemistry 2015, 54, 4297–4306. [CrossRef] [PubMed] 40. Li, M.; Zhao, C.; Yang, X.; Ren, J.; Xu, C.; Qu, X. In Situ Monitoring Alzheimer’s Disease β-Amyloid Aggregation and Screening of Aβ Inhibitors Using a Perylene Probe. Small 2013, 9, 52–55. [CrossRef] 41. Hasecke, F.; Miti, T.; Perez, C.; Barton, J.; Schölzel, D.; Gremer, L.; Grüning, C.S.R.; Matthews, G.; Meisl, G.; Knowles, T.P.J.; et al. Origin of metastable oligomers and their effects on amyloid fibril self-assembly. Chem. Sci. 2018, 9, 5937–5948. [CrossRef] 42. Miti, T.; Mulaj, M.; Schmit, J.D.; Muschol, M. Stable, Metastable and Kinetically Trapped Amyloid Aggregate Phases. Biomacromolecules 2015, 16, 326–335. [CrossRef] 43. Soreghan, B.; Kosmoski, J.; Glabe, C. Surfactant properties of Alzheimer’s A beta peptides and the mechanism of amyloid aggregation. J. Biol. Chem. 1994, 269, 28551–28554. 44. Sabaté, R.; Estelrich, J. Evidence of the Existence of Micelles in the Fibrillogenesis of β-Amyloid Peptide. J. Phys. Chem. B 2005, 109, 11027–11032. [CrossRef] 45. Nick, M.; Wu, Y.; Schmidt, N.W.; Prusiner, S.B.; Stöhr, J.; DeGrado, W.F. A long-lived Aβ oligomer resistant to fibrillization. Biopolymers 2018, 109, e23096. [CrossRef] 46. Hill, S.E.; Miti, T.; Richmond, T.; Muschol, M. Spatial Extent of Charge Repulsion Regulates Assembly Pathways for Lysozyme Amyloid Fibrils. PLoS ONE 2011, 6, e18171. [CrossRef] 47. Zandomeneghi, G.; Krebs, M.R.H.; McCammon, M.G.; Fändrich, M. FTIR reveals structural differences between native β-sheet proteins and amyloid fibrils. Protein Sci. 2004, 13, 3314–3321. [CrossRef] 48. Laganowsky, A.; Liu, C.; Sawaya, M.R.; Whitelegge, J.P.; Park, J.; Zhao, M.; Pensalfini, A.; Soriaga, A.B.; Landau, M.; Teng, P.K.; et al. Atomic View of a Toxic Amyloid Small Oligomer. Science 2012, 335, 1228–1231. [CrossRef] 49. Vieira, M.N.N.; Forny-Germano, L.; Saraiva, L.M.; Sebollela, A.; Martinez, A.M.B.; Houzel, J.-C.; de Felice, F.G.; Ferreira, S.T. Soluble oligomers from a non-disease related protein mimic Aβ-induced tau hyperphosphorylation and neurodegeneration. J. Neurochem. 2007, 103, 736–748. [CrossRef] 50. Parmar, A.S.; Gottschall, P.E.; Muschol, M. Sub-micron Lysozyme Clusters Distort Kinetics of Crystal Nucleation in Supersaturated Lysozyme Solutions. Biophys. Chem. 2007, 129, 224–234. [CrossRef] 51. Ghosh, P.; Kumar, A.; Datta, B.; Rangachari, V. Dynamics of protofibril elongation and association involved in Aβ42 aggregation in Alzheimer’s disease. BMC Bioinform. 2010, 11, S6–S24. [CrossRef] 52. Al-Hilaly, Y.K.; Williams, T.L.; Stewart-Parker, M.; Ford, L.; Skaria, E.; Cole, M.; Bucher, W.G.; Morris, K.L.; Sada, A.A.; Thorpe, J.R.; et al. A central role for dityrosine crosslinking of amyloid-β in Alzheimer’s disease. Acta Neuropathol. Commun. 2013, 1, 83–100. [CrossRef] 53. Buell, A.K.; Dobson, C.M.; Knowles, T.P.J.; Welland, M.E. Interactions between Amyloidophilic Dyes and Their Relevance to Studies of Amyloid Inhibitors. Biophys. J. 2010, 99, 3492–3497. [CrossRef] Biomolecules 2019, 9, 539 15 of 15

54. Jahn, T.R.; Radford, S.E. Folding versus aggregation: Polypeptide conformations on competing pathways. Arch. Biochem. Biophys. 2008, 469, 100–117. [CrossRef] 55. Lee, J.; Culyba, E.K.; Powers, E.T.; Kelly, J.W. Amyloid-β forms fibrils by nucleated conformational conversion of oligomers. Nat. Chem. Biol. 2011, 7, 602–609. [CrossRef] 56. Fu, Z.; Aucoin, D.; Davis, J.; van Nostrand, W.E.; Smith, S.O. Mechanism of Nucleated Conformational Conversion of Aβ42. Biochemistry 2015, 54, 4197–4207. [CrossRef] 57. Hackl, E.V.; Darkwah, J.; Smith, G.; Ermolina, I. Effect of acidic and basic pH on Thioflavin T absorbance and fluorescence. Eur. Biophys. J. 2015, 44, 249–261. [CrossRef] 58. Fodera, V.; Groenning, M.; Vetri, V.; Librizzi, F.; Spagnolo, S.; Cornett, C.; Olsen, L.; van de Weert, M.; Leone, M. Thioflavin T Hydroxylation at Basic pH and Its Effect on Amyloid Fibril Detection. J. Phys. Chem. B 2008, 112, 15174–15181. [CrossRef]

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