To explore possible effects of the htt exon1 N-terminal 17 amino acids on polyQ aggregation, we generated the peptide htt^NTQ₃₅ (Table 1), subjected it to a disaggregation procedure required to ensure the absence of preexisting aggregates 24, and studied its aggregation in PBS buffer at 37 °C. We found that 3 μM htt^NTQ₃₅ undergoes aggregation significantly (p < 0.001 at t = 10.5 h) more rapidly than 3 μM Q₃₅ (Fig. 1A). In addition, the peptide htt^NTQ₃₆P₁₀ aggregates somewhat less rapidly than htt^NTQ₃₅ but much more rapidly than Q₃₅ (Fig. 1A); this shows that while the aggregation suppressing ability of oligoPro also operates within the exon1 context, the enhancing effect of htt^NT is dominant over the suppressing effect of oligoPro. As discussed above, oligoPro placed C-terminal to polyQ diminishes aggregation rate compared with polyQ (compare 25 μM Q₃₅P₁₀ with 25 μM Q₃₅ in Fig. 1A), but placed N-terminal to polyQ has no effect (not shown) 14. In contrast, the htt^NT effect does not appear to depend on where the htt^NT is placed. Thus, Q₃₅htt^NT exhibits an aggregation rate that is much faster than that of Q₃₅ and not significantly (p > 0.01 at 0.75 h) different to that of htt^NTQ₃₅ (Fig. 1A).

In spite of the apparent dominant effect by the htt^NT sequence, the kinetics of a series of htt^NTQ_N peptides continues to exhibit a strong polyQ repeat length dependence, as shown previously for both simple polyQ peptides 25 and for recombinantly produced exon1 peptides 9. Thus, while htt^NT, htt^NTQ₃, and htt^NTQ₁₅ all aggregate sluggishly, htt^NTQ₂₅ aggregates over a period of 1−2 days and, as discussed above, htt^NTQ₃₅ aggregates within a few hours (Fig. 1B). Although some of these peptides examined in Figure 1B contain a Phe17->Trp mutation to allow certain fluorescence experiments (described below), this mutation does not appreciably affect aggregation properties. Control experiments in an htt^NTQ₂₀P₁₀ background show that replacement of either Phe₁₁ or Phe₁₇ with Trp in the htt^NT segment yields aggregation kinetics very similar to those of the corresponding wild type peptide (Fig. 1C).

Previously, we found that early stages of the aggregation of simple polyQ peptides exhibit a modest concentration dependence consistent with a nucleated growth polymerization mechanism and a critical nucleus of one 5, as shown for Q₃₀ in Figure 1D. In contrast, the initial stages of aggregation of the exon1 related sequence htt^NTQ₃₀P₆ (previously we found no appreciable difference between a Pro₆ and a Pro₁₀ sequence 14) yields a log-log plot of initial kinetics vs. concentration (Fig. 1D) 5 with a slope of approximately 1, corresponding to a calculated critical nucleus (n*) of about −1. The n* = −1 value indicates a rate - concentration relationship for the early aggregation kinetics of htt^NTQ₃₀P₆ that is consistent with a nonnucleated, “downhill” aggregation mechanism 26 for oligomer formation, without a kinetic barrier to spontaneous aggregation. This analysis suggests that polyQ peptides containing the htt^NT sequence spontaneously aggregate by an entirely different mechanism than simple polyQ peptides, a conclusion that is further supported by additional data presented below.

Previously, we found that when simple polyQ monomers undergo spontaneous aggregation in aqueous solution, the earliest observable aggregates have fibril-associated properties similar to those of the final product 5. In contrast, electron micrographs show that the initial products in the aggregation of htt^NTQ₃₀P₆ are oligomeric and protofibrillar (Fig. 2). Later in the time course, various other aggregated structures appear that are more fibril-like (Fig. 2). A relationship between the formation of such oligomeric products and non-nucleated, downhill aggregation kinetics (Fig. 1D) fitting a classical colloidal coagulation model has been suggested previously for other protein aggregation reactions 27,28.

There are a number of possible explanations for the aggregation-enhancing ability of the htt^NT sequence. (I.) Since addition of lysine residues to a sequence sometimes discourage aggregation 29, htt^NT might enhance exon1 aggregation compared to our simple polyQ peptides because it replaces the Lys-Lys pair at the N-terminus of the model polyQ peptides (see introduction and Table 1). However, the aggregation kinetics of 3 μM htt^NTK₂Q₃₅, an exon1 analog containing a Lys-Lys pair inserted between htt^NT and Q₃₅, are not significantly (p > 0.01 at 0.3 h) different from the kinetics of 3 μM htt^NTQ₃₅ while at the same time are significantly (p < 0.001 at t = 10.5 h) faster than the kinetics of 3 μM Q₃₅ (Fig. 1A). In addition, mutations that reduce hydrophobic character of htt^NT without altering charged residues abrogate the rapid aggregation kinetics in an exon1 peptide (Fig. 1C), confirming that the htt^NT segment provides some positive, sequence-specific contribution to aggregation, rather than simply replacing a Lys-Lys aggregation suppressor. (II.) An obvious alternative possibility is that htt^NT itself might be highly aggregation-prone. Surprisingly, however, we found that a peptide consisting solely of the htt^NT sequence aggregates sluggishly under our standard conditions (Figs. 1A, 1B). (III.) Another possible explanation is that the combination of all or part of htt^NT with the first portion of the polyQ sequence might create a new aggregation sequence motif with much more robust aggregation kinetics than either polyQ or htt^NT alone. However, htt^NT peptides containing short to intermediate lengths of polyQ also aggregate very slowly (Fig. 1B), and robust aggregation kinetics ensue for longer polyQ constructs even when htt^NT is attached to polyQ C-terminus (Fig. 1A), which creates an entirely different sequence at the Q_n-htt^NT junction; these data suggest that polyQ abutted to htt^NT does not simply create a powerful, novel, linear aggregation motif. The htt^NTK₂Q₃₅ data (Fig. 1A) also argues against a novel aggregation motif, since if there were such a motif, at the junction of the htt^NT and polyQ sequences, the highly charged Lys-Lys insertion would be expected to disrupt it. (IV.) It is also possible that the htt^NT sequence might normally exist as a well-behaved oligomeric species, such as a dimer or trimer, which could accelerate aggregation by concentrating and/or orientating the polyQ elements. However, size exclusion chromatography (SEC) mobility shows that the htt^NT peptide migrates as a monomer (Fig. 3), while SEC elution profiles (not shown) show no evidence of higher assembly states. Likewise, circular dichroism (CD) spectra of htt^NT do not change with respect to concentration (Fig. 4A), consistent with a non-associating system. (V.) Another possibility is that, in analogy to a recent report of the ability of expanded polyQ to destabilize a folded protein domain 20, the htt^NT segment may exist in a folded and/or compact state that resists aggregation, but unfolds or extends when attached to expanded polyQ, enhancing its aggregation; disruption of the folded state is a common trigger for globular protein aggregation 30. This last hypothesis was explored in detail, as described next.

Arguing against this postulated polyQ-induced unfolding mechanism for the htt^NT effect is the fact that most peptides the size of htt^NT do not fold into stable, globular structures, but rather are disordered. Interestingly, however, analytical SEC suggests that the htt^NT sequence is actually relatively compact in solution. A series of simple polyQ peptides yield migration rates in SEC that fit a straight line (Fig. 3A). As expected, peptides that are predicted to be extended and thus have larger hydrodynamic radii, such as an Ala-rich peptide strongly favoring α-helix 31, and a Pro₁₄ peptide favoring polyproline type II helix 32, migrate faster than equivalent length polyQ peptides. Insulin and aprotinin, which exhibit smaller hydrodynamic radii caused by their compact structures, migrate – as expected - more slowly than the equivalent length polyQ peptides. Within this dataset, the htt^NT peptide also migrates significantly more slowly for a peptide of its molecular weight, suggesting that– like insulin and aprotinin – htt^NT has a relatively small hydrodynamic radius.

To confirm the compactness of the htt^NT peptide, we conducted a fluorescence-based resonance energy transfer (FRET) experiment in which we replaced Met₁ of htt^NT with the resonance energy acceptor, nitro-Tyr, and Phe₁₇ with the resonance energy donor Trp 33, in the context of an htt^NTQ₃ sequence (“FRET-htt^NTQ₃”). Compared with htt^NTQ₃ containing only the F17W replacement, the Trp fluorescence of the FRET-htt^NTQ₃ peptide decreases by about 50% (data not shown), corresponding to a calculated average separation between donor and acceptor groups of 24 ± 0.5 Å (Fig. 3B). This is significantly shorter than the average end-to-end distance of 34.5 ± 4 Å calculated 34 for a 17-residue peptide in an extended statistical coil conformation. This theoretical value is supported by a FRET analysis of the htt^NTQ₃ peptide in 6 M urea, which yields a separation of 33.9 ± 0.5 Å (Fig. 3B). Thus, in agreement with the SEC data, htt^NT in the htt^NTQ₃ context in native buffer appears to be significantly collapsed.

Given this evidence for a collapsed structure in an isolated htt^NT peptide, we investigated whether it was possible for expanded polyQ sequences to disrupt that collapsed state in peptides dissolved in PBS. We found that for the FRET-exon1 mimic peptide containing a Q₂₀ repeat, the average separation between nitro-Tyr and Trp in the htt^NT sequence does not significantly change (p > 0.01). However, the corresponding separation for the Q₃₇ repeat peptide expands to 32 ± 1 Å (p < 0.01), a value approaching the 34 Å range obtained both by measurement of htt^NTQ₃ in urea and by calculation based on an assumed statistical coil configuration (Fig. 3B). The data is thus consistent with a mechanism in which the htt^NT segment of exon1 is normally in a collapsed state that is resistant to aggregation, but that when connected to an expanded polyQ sequence becomes extended and labile to aggregation - an effect analogous to what has previously been observed for a globular protein 20.

Although several features of the above results are consistent with htt^NT (in the absence of a connected expanded polyQ) being a compact domain, the short length of htt^NT makes it unlikely (but not impossible) that it possesses a unique, folded structure. To probe htt^NT secondary and tertiary structure, we applied two solution methods. The first, CD spectral analysis, proved equivocal. The CD spectrum of htt^NT at 35 °C (Fig. 4A) and the 35 °C – 5 °C difference spectrum (not shown) lack strong secondary structure features, suggesting the absence of a stable structure. At the same time, deconvolution analysis of the 35 °C spectrum predicts significant α-helix (see legend, Fig. 4), consistent with projections based on amino acid sequence 23,35. This interpretation of the CD spectrum is problematic, however, since the protein datasets used in programs like ContinLL are thought to be not suitable for deconvoluting CD spectra of short peptides 36.

The CD dichotomy was clarified by high resolution NMR analysis (Fig. 5), which strongly suggests the absence of stably folded structure. Two-dimensional proton TOCSY and NOESY NMR analyses show that htt^NT adopts predominantly unfolded, random-coil conformations, characterized by small spectral dispersion, small secondary chemical shifts, and strong sequential H^α(i)-H^N(i+1) NOEs with few sequential H^N(i)-H^N(i+1) or medium-range NOEs. A single, very weak, medium-range NOE cross-peak is observed connecting the Thr3 H^α and Glu5 HN protons (Fig. 5B). The region around these residues shows the largest negative H^α secondary chemical shifts and exhibits the most H^N(i)-H^N(i+1) NOE cross-peaks (Fig. 5A), which indicates transient existence of a few residues in the α-helix quadrant of Φ,Ψ space. Despite this slight propensity, there is clearly no stable α-helix in this peptide in solution under physiological conditions.

Thus, in the absence of an expanded polyQ, htt^NT adopts a conformation lacking significant secondary and tertiary structural features, while at the same time being in a collapsed state. Results from the sequence analysis algorithm PONDR are consistent with this, predicting (Fig. 6) the htt^NT sequence to be a molecular recognition feature (MoRF) 37 capable of engaging in coupled folding and binding interactions 15. Such sequences tend to be collapsed coils in native buffer 15, often taking up α-helical conformations when complexed with their binding partners. Consistent with this, htt^NT has essentially no α-helix in isolation (see above) but takes on significant α-helix in the presence of low concentrations of trifluoroethanol as revealed by CD (Fig. 4B).

When the assembly of simple polyQ peptides into amyloid-like aggregates is monitored by multiple analytical techniques, the data from all measures track closely, suggesting the absence of reaction intermediates 5. In contrast, htt^NT-containing exon1 peptides exhibits different aggregation curves depending on the analytical method (Figs. 7 and 8). In particular, thioflavin T binding (Δ), commonly used for measuring amyloid-like structure 38, generates reaction curves that are delayed compared to the curves from the HPLC sedimentation (◆) and light scattering (○) assays (Fig. 7B, 8C). Thus, for the htt^NTQ₃₀P₆ peptide (Fig. 7) at 6.25 hrs, the difference between the ThT value (Δ) and the HPLC value (◆) is statistically significant (p < 0.01); at later time points (19 hrs, 120 hrs) the difference is insignificant (p > 0.01). The ThT lag followed by a burst suggests that the initial aggregates are not amyloid-like, while later aggregates are. FTIR analysis also suggests that the initial aggregation product is qualitatively different - exhibiting more coil and less β-structure - compared to aggregates isolated later in the time course (Fig. 8E). This is also consistent with the EM data (Fig. 2).

Details of the structures of these intermediates are revealed by further analysis of htt^NTQ₃₀P₆ (Fig. 7) and htt^NTQ₂₀P₁₀ (Fig. 8) aggregates isolated from ongoing reactions. In particular, dot blot analysis of these aggregates show that polyQ in the initial aggregates is readily accessible to an antibody against a linear polyQ epitope, but is masked in the later aggregates (Figs. 7C, 8D). In addition, the initially formed aggregates are poor templates for recruiting polyQ-containing peptides, while the later, fibrillar aggregates are capable of seeding polyQ elongation (Fig. 8B). Seeding activity is a feature particularly associated with amyloid-like aggregates 39. Furthermore, while a Trp residue inserted for Phe at position 11 or 17 is solvent exposed both in the monomeric peptide and in the initial aggregates, it becomes less solvent accessible (based on its fluorescence maximum) as the aggregation reaction proceeds, with a time course that parallels the disappearance of polyQ antibody binding epitopes (Figs. 7A (■); 8B (•,▲)). In contrast, when the isolated htt^NT peptide itself slowly aggregates, the Trp residue of the F17W mutant remains fully solvent exposed in the aggregate fraction even after 800 hrs (Fig. 7A (Δ)); this is consistent with the apparently inability of aggregates of htt^NT lacking a long polyQ track to progress beyond the oligomer stage, in agreement with EM analysis (Fig. 2L). While the initial aggregates of htt^NTQ_N peptides appear to be less tightly packed than the later aggregates, they are sufficiently structured that they are protected from proteolyis by trypsin, in contrast to soluble, monomeric htt^NT (Fig. 8A).

Thus, the initial aggregates in this htt^NT mediated mechanism are non-fibrillar oligomers with their htt^NT segments composing the core, but their polyQ segments remaining unstructured and available for antibody binding. Subsequently, the polyQ elements also become integrated into the aggregate core structure, which takes on a more fibrillar character both in the polyQ and htt^NT elements. Growth into larger fibrillar assemblies, as seen in EM, take place at later incubation times. FTIR analysis (Fig. 8E) shows that the initial aggregates (before burial of Trp₁₇) contain a significant amount of coil and turn conformations in addition to β-structure. Interestingly, FTIR spectra of all aggregates isolated after Trp₁₇ burial feature a single β-sheet band at ∼1,626 cm⁻¹ and are indistinguishable from each other and from aggregates of simple polyQ (Fig. 8E).

Critically, at a time when the major changes in aggregate structure are occurring, as evidenced by ThT binding, Trp fluorescence shift, FTIR, EM, and polyQ antibody binding, the vast bulk of the exon1 peptide (>80%) does not pellet after centrifugation (Figs. 7 and 8) and migrates as a single, monomeric (Fig. 3A) species in size exclusion chromatography (M. Jayaraman and R. Wetzel, unpublished data). Coincident with the timing of the changes in aggregate structure, there is a dramatic increase in the rate of monomer loss, suggesting that a nucleation event has occurred.

All huntingtin-related peptides, as well the Pro14 and Ala-rich peptide “Bal” 31, were obtained in non-purified form from the small-scale custom peptide synthesis facility of the Keck Biotechnology Center at Yale University (http://keck.med.yale.edu/ssps/). Peptides were purified by reverse phase HPLC and structures confirmed by mass spectroscopy on an Agilent 1100 electrospray MSD 24. Purified peptides were routinely freshly disaggregated before use, as described 24. Porcine insulin and aprotinin were from Sigma-Aldrich. Acetonitrile, hexafluoroisopropanol (99.5% (v/v), spectrophotometric grade), and formic acid were from Acros Organics, trifluoroacetic acid (99.5% (v/v), Sequanal Grade) was from Pierce, and trifluoroethanol was from Sigma-Aldrich.

The sedimentation assay 24, the ThT and 90° light scattering assays 51, and the nucleation kinetics analysis 5,24, have been described. The isolation of aggregates for analysis was conducted by centrifuging a reaction aliquot at 20,817 × g in an Eppendorf centrifuge at 4 ° C for 30 mins, washing the pellet 2−3 times with PBS, resuspending in buffer, and determining the aggregate concentration by an HPLC analysis of a dissolved aliquot, as described 24.

Aliquots of whole aggregation reaction mixture were taken at different time points and visualized by electron microscopy. 3 μl of sample were placed on a freshly glow-discharged carbon-coated grid, adsorbed for 2 minutes, washed with deionized water before staining with 2μl of 1% uranyl acetate and blotting. Grids were imaged on a Tecnai T12 microscope (FEI Co., Hillsboro, Oregon) operating at 120kV and 30,000× magnification, and equipped with an UltraScan 1000 CCD camera (Gatan, Pleasanton, California) with post-column magnification of 1.4×.

CD spectra were collected using a Jasco J-810 spectrapolarimeter with a 1 mm pathlength quartz cuvette. Htt^NT samples were collected in 20 mM Tris-TFA pH 7.2 at concentrations of 20 − 500 μM. The spectra were collected immediately after thawing (from −80 °C) the disaggregated peptide samples. Scans were made at 20 nm per min with steps of 0.5 nm and an averaging time of 8s. The reported spectra are averages taken over 4 scans.

The htt^NT sample for NMR experiments contained 40 μM peptide in 20mM sodium phosphate buffer (pH 7.2), 0.02 % sodium azide, 6% ²H₂O. NMR experiments were carried out on a Bruker Avance 800 MHz NMR spectrometer, equipped with a 5 mm z-axis gradient cryoprobe. The water solvent peak was suppressed using the WATERGATE W5 pulse sequence 52. Two dimensional (2D) homonuclear NOESY and TOCSY 53 data were acquired at 5 °C, using a 1.5 s recycle delay and mixing times of 200 ms and 60 ms. Complete backbone and side-chain proton assignments of htt^NT were obtained. H^α secondary chemical shifts (Δδ H^α) were calculated by subtracting sequence-corrected random coil values 54.

Size exclusion chromatography experiments were conducted with a Superdex peptide HR10/30 (Pharmacia Biotech) column on a Bio-Rad (Biologic Duo flow) chromatograph using 1ml min⁻¹ flow rate and detection at 215 nm at ambient temperature. Peptides were suspended in PBS (polyQ peptides and htt^NT after disaggregation) at 20−100 μM and 100 μL injected. The Kav values and the plot between Kav vs. logMW were calculated as described in the GE Healthcare handbook Gel Filtration: Principles and Methods available on the GE Healthcare web site.

FRET peptides (Table 1) were purified and disaggregated. The peptide solution in TFA/water, pH 3.0 obtained after the ultracentrifugation step of the disaggregation procedure was immediately adjusted to 5 μM concentration in 10 mM PBS, pH 7.4 and its fluorescence determined. Peptide solutions were confirmed to be aggregate-free at the end of the FRET measurements by electron microscopy, HPLC, ThT, and 90° light scattering measurements. Fluorescence spectra were recorded at room temperature with excitation and emission slit widths at 5 nm. Raw data obtained were buffer subtracted and energy transfer efficiencies (E) were calculated from fluorescence intensities at 353.5 nm from the emission spectra of the F17W and doubly labeled FRET peptide. The D-A distance (r) for each peptide was determined from the E value and the Ro value (26 Å) for the nitrotyrosine / tryptophan FRET pair 33,55. The measurements were carried out in triplicate and the mean value (r) is reported with standard deviation.

Peptide concentrations underpinning the FRET determination were made in two different ways, which gave very similar results. In one method, we used amino acid composition analysis (Keck Biotechnology Center, Yale University) to calibrate a stock solution of the peptide which was then used as an HPLC standard for future determinations 24. In the second method, the UV absorption peaks at 280 nm of Trp and 381 nm of nitro tyrosine were used to normalize concentrations of F17W and doubly labeled FRET peptides using the extinction coefficients of Trp ε_{280 nm} = 5,600 M⁻¹ cm⁻¹ and nitro tyrosine ε_{381 nm} = 2,200 M⁻¹ cm⁻¹ , respectively 56.

Dot blots were performed as described 21. Aggregate samples were harvested, resuspended, and quantified as described in General Methods, and aliquots containing 400 ng of aggregates were transferred to a nitrocellulose membrane. In parallel, a portion of the unfractionated aggregation reaction mixture was transferred in to nitrocellulose membrane at various time intervals. Blots were incubated with Tris-buffered saline (TBST) containing 5% BSA overnight, washed three times with TBST, and incubated with a 10 nM solution of purified MW1 antibody 57 (a gift from Jan Ko and Paul Patterson) for 2 h. After washing with TBST to remove unbound material, blots were incubated 2 hrs with a 1:15,000 dilution of a peroxidase conjugate of anti-mouse IgG (whole molecule) (Sigma, A4416), then washed four times with TBST. Blots were visualized with enhanced chemiluminescence solution (Pierce #A4416) following the manufacturer's instructions.

Aggregates were isolated at different times as described in General Methods, then resuspended in 300 μL 150 mM NaCl, 10 mM phosphate, pH 7.4 and analyzed on a Perkin Elmer luminescence LS50B spectrometer. The samples were excited at 280 nm and emission was scanned between 290 and 550 nm. The wavelength at the emission intensity maximum was recorded.

Fourier transform infrared spectroscopy of various samples was performed using MB series spectrophotometer with PROTA software (ABB Bomem). Protein aggregates were harvested by centrifugation at 20,817 × g and the pellet was washed 3 times with PBS. Spectra of re-suspended aggregates were recorded at 4 cm⁻¹ resolution (400 scans at room temperature). Spectra were corrected for the residual buffer absorption by subtracting the buffer alone spectrum interactively until a flat baseline was obtained between 1,700−1,800 cm⁻¹. Second-derivative spectra for the Amide I region were calculated from the primary spectrum by using PROTA software.

For the Q₁₅ monomer sample, the peptide was purified by reverse phase HPLC with an aqueous acetonitrile gradient in 5 mM HCl to avoid exposure of the peptide to TFA, which gives a large peak in FTIR. Peak fractions were pooled and lyophilized, and the powder dissolved in 50 μl of 1 mM HCl , then centrifuged 1 hr at 435,680 × g. A 25 μl aliquot was carefully removed and mixed with 25 μL of a 2X PBS buffer, centrifuged for 30 mins at 435,680 × g, and the supernatant subjected to analysis. This material appears to contain about 20% by mass of amyloid-like polyQ aggregates, based on ThT analysis, presumably because of its very high concentration (2.4 mM) and the abbreviated and modified disaggregation protocol necessitated by the demands of the experiment.

For the monomer control, htt^NT was disaggregated and dissolved in pH 3 TFA in water, then added to a solution of trypsin (SEQUENZ-Trypsin, Worthington Biochemical Corporation) in 100 mM Tris-HCl, pH 7.0 to yield a 1:10 ratio of trypsin to peptide in 50 mM Tris. This solution was incubated at room temperature and monitored by injection of aliquots onto a RP-HPLC-MS system (Agilent 1100), which indicated efficient cleavage after the htt^NT Lys residues (data not shown). Aggregates were harvested and quantified as described in General Methods, then incubated with trypsin at 1:10 w/w (trypsin: peptide) in 50 mM Tris, pH 7.0 at room temperature. LC-MS of digest centrifugation supernatants yielded no material. All the material was found in pellet fractions and was undigested (data not shown).

The elongation of biotinylated Q₂₉ (B-Q₂₉) on htt^NTQ₂₀P₁₀ aggregates harvested at different times was done as described 6.

For all reaction profiles, datasets were fit in Sigma Plot to either 3-parameter equations (exponential decay, exponential rise to maximum, or sigmoidal) or linear regression. Reported R² values and standard deviations (“S.D.”) are from the Sigma Plot fits. Many data sets were obtained in duplicate, and those that were not are representative of multiple experiments. The tight error bars for the HPLC sedimentation assay data obtained in duplicate (Fig. 1A, all datasets but for htt^NT and htt^NTQ₃₆P₁₀), along with the excellent curve fits throughout Figure 1, give us confidence in the datasets where only single replicates were taken (Figs. 1B and 1C). Duplicate data points in Figure 7 were derived from two experiments run at different times, and some points (0.6 hrs, 4 hrs: ThT, LS; 28 hrs, 69 hrs: HPLC, ThT, LS) are represented by only one replicate. The larger error bars for ThT and LS readings at the later time points are typical for late-stage amyloid formation reactions as fibrils grow larger. P-values were calculated using the two-tailed Student's t-test.