Chimera-Induced Folding: Implications for Amyloidosis

Article pubs.acs.org/Biomac

Chimera-Induced Folding: Implications for Amyloidosis Gaius A. Takor,† Seiichiro Higashiya,† Mirco Sorci,‡ Natalya I. Topilina,§ Georges Belfort,‡ and John T. Welch*,† †

Department of Chemistry and §Department of Biological Sciences, University at Albany, State University of New York, Albany, New York 12222, United States ‡ Howard P. Isermann Department of Chemical and Biological Engineering and The Center for Biotechnology and Interdisciplinary Studies Rensselaer Polytechnic Institute, Troy, New York 12180, United States ABSTRACT: The discoveries that non-native proteins have a role in amyloidosis and that multiple protein misfolding diseases can occur concurrently suggest that cross-seeding of amyloidogenic proteins may be central to misfolding. To study this process, a synthetic chimeric amyloidogenic protein (YEHK21-YE8) composed of two components, one that readily folds to form fibrils (YEHK21) and one that does not (YE8), was designed. Secondary structural conformational changes during YEHK21-YE8 aggregation demonstrate that, under the appropriate conditions, YEHK21 is able to induce fibril formation of YE8. The unambiguous demonstration of the induction of folding and fibrillation within a single molecule illuminates the factors controlling this process and hence suggests the importance of those factors in amyloidogenic diseases.

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INTRODUCTION Ordered protein aggregates, especially amyloid fibrils, are central to the pathogenic events in protein misfolding diseases (PMDs) including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and transmissible spongiform encephalopathy (TSE).1−3 Ordered aggregation also occurs in type II diabetes, in chronic inflammatory conditions and in many skeletal muscle disorders. A hallmark event of PMDs is the change in the secondary and tertiary structure of a native protein without alteration of the primary structure. Common structural changes include increases in βsheet content, in oligomerization and in formation of fibrillar amyloid-like polymers.3 The kinetics of fibrillation and the relationship between fibrillation and toxicity have been studied in great detail for insulin.4−6 In particular, the pH dependence of β-strand formation has been compared and contrasted with the role of insulin seeds on fibrillation. Amyloid fibrils are normally homogeneous, composed of a single protein or peptide. Consistent with that observation, when the B chain of insulin is mixed with insulin at pH 7.5 no heterogeneous fibrils are formed, however at pH 1.6, heterogeneous fibrils do form.7 The relevance of these findings to the interactions of a chimeric construct will be described in this work. The kinetics of amyloid fibril formation and the toxicity of those fibrils have received considerable attention.8,9 The precursor oligomers appear to be the etiologic agents of toxicity where fibril formation serves as a natural storage or detoxification mechanism for the cell.10 Two types of amyloid fibrils may be formed in a cell, in vivo formed pathogenic amyloid and functional amyloid.11 Compelling in vitro experiments suggest that fibrillation begins with a lag phase © 2014 American Chemical Society

forming reactive nuclei (nucleation step) followed by a selftemplated growth phase and finally the saturation phase.12−14 Fibrillation in vitro can be forced by extremes of temperature or denaturant concentration; however, the majority of naturally occurring amyloidogenic proteins can form fibrils under mild conditions. It requires the formation of partially structured forms that facilitate intermolecular contacts, however there is no consensus on the nature of the elongation step or on the process of the formation of multistranded fibrils.6 The lag phase associated with fibrillation is typically attributed to the growth of the fibrillar nucleus,15,16 with the formed protofilament driving the elongation phase. Multiple prefibrillar stages are involved with the consequence that the stability of prefibrillar aggegrates is highly variable. Seeding with preformed fibril seeds can bypass the required nucleation step.5,17 The necessary seeds can originate by fragmentation of extant fibrils.18−20 The formation of protein aggregates can be rationalized to a considerable extent by simple physicochemical parameters,21 such as pH, temperature, ionic strength, and concentration.15,22−26 Intrinsic factors associated with amyloid formation include polypeptide characteristics, such as charge,27−29 hydrophobicity,30−32 patterns of polar and nonpolar residues,33 and the propensities of primary sequences to adopt diverse secondary structural motifs.28,32,34,35 Charge has been shown to contribute to the aggregation rate of a polypeptide chain in a manner inversely proportional to the absolute value of the net charge. The rate of aggregation may be positively correlated with pH. In particular, it has been observed that in vitro Received: April 24, 2014 Revised: July 1, 2014 Published: July 8, 2014 2992

dx.doi.org/10.1021/bm5006068 | Biomacromolecules 2014, 15, 2992−3001

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amyloid fibril formation often occurs at low pH.25,33,36−38 Charged amino acids in the polypeptide sequence can promote specific repulsive39 or attractive interactions.40 We have previously shown that the presence of charge is not necessarily as relevant as the distribution of that charge.41 Solvent properties including polarity and hydrogen bonding strength can be used to control and tune fibril formation.4,42,43 The effect of interface chemistry and osmolytes in solution on amyloidosis kinetics has also been reported.4 Hydrophobicity appears to be a common thread inducing amyloidosis. Elongation has been suggested to occur by growth from either or both ends of a fibril, but it is also possible for oligomers to join directly for the lateral association of fibrils.6,44 Although the concept of structural heredity is nearly 50 years old,45 the crucial understanding of the mechanism of fibril transduction, identification of the point in the folding process where fibrillation become self-propagating,46 remains to be elucidated. There is an unproven hypothesis that amyloid fibril formation promoted by self-templating peptides containing cross β-strands47 is sufficient to induce fibrillation. It is proposed that terminal strands capture native proteins and redirect folding. Amyloidogenesis is typically a specific process for self-seeding where primary sequence mismatches disrupt folding, consequently cross seeding is not normally effective. Sequence based barriers to cross seeding can be bypassed48 by seeding with heterologous fibrils even though point mutations can drastically alter cross seeding capacity. Unfortunately, the mechanism of formation and the structural characterization of the prefibrillar species is lacking.49 The simultaneous presentation of AD and TSE in patients (Aβ and prion protein coexisting) has been reported widely.50−52 The finding that an amyloidogenic protein and etiologic agent of one disease state is a risk factor for another amyloid disease is suggestive of significant interactions between various amyloid proteins. The small molecular chaperone heat shock protein, αB-crystallin (αB), binds Aβ in plaques found in AD patients and contributes to the neurotoxicity and metabolism of Aβ. However, the nature of the interaction between Aβ and αB and toxicity effects is unclear, although hydrophobic interactions may be operative. The majority of type 2 diabetes patients have pancreatic amyloid deposits composed of islet amyloid polypeptide. Islet amyloid polypeptide shares a striking primary sequence similarity with Aβ.53,54 Despite amino acid sequence differences, cross-seeding has also been shown to occur between wild Aβ and hereditary Aβ variants, including Dutch- and Flemishtypes Aβ proteins and between some yeast prions.55,56 These results fueled interest in cross seeding between amyloid proteins.57−59 Even though cross-seeding can occur when there is a striking similarity in the primary sequences of the distinct polypeptides, secondary and tertiary structural similarities rather than sequence homology is considered critical to the process.60,61 The interspecies barrier is believed to be mediated by physical interactions, such as specific packing of the amino acid side chains within the self-propagating cross-β-structures.62 Energetically favorable formation of the cross β core can compensate for primary sequence mismatches. These studies illuminate the feasibility of promiscuous cross seeding in amyloidosis, yet the role of charge distribution in cross seeding is not clear. The natively disordered aggregates implicated in the aforementioned PMDs have significant net charge at physiological pH and yet form amyloid fibrils in the

disease state.63 To investigate the role of net charge and charge distribution in cross-seeding, a chimeric polypeptide, GH 6 [(GA) 3 GY(GA) 3 GE(GA) 3 GH(GA) 3 GK] 21 [(GA) 3 GY(GA)3GE]8GAH6, YEHK21-YE8, based on the previously prepared YEHK2164 and YE865 constructs was designed, expressed, and purified. YEHK21, YE8, and the chimeric construct, YEHK21-YE8, were designed to form β-sheet structures with the β-turns decorated by specific amino acids (Figure 1).

Figure 1. Representative units of polypeptides: (A) 2 repeats of YE8; (B) 1 repeat of YEHK21; and (C) YEHK21-YE8 showing 21st repeat of YEHK21 block seamlessly linked to the YE8 block with 2 repeats shown.

Both YEHK21 and YE8 form identical GAGAGA β-strands. Previously YEHK21 was shown to rapidly form β-sheet assemblages (refolds in ∼1 h after melting) that condense into well-defined fibrillar structures on incubation at pH 6.5.41,64,66 However, at pH 6.5 YE8 does not form fibrils. Only following incubation at pH 3.5 for a longer period (∼45 days) does YE8 form well-defined fibrillar structures.41,67 In a 2993


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was added) to a final volume of 4 mL (0.5% templating material). In tube B, 20 μL of 44 μM solution of fibrillated and then sonicated YEHK21 was added and YEHK21-YE8 seed was added in an equal portion to tube C. As controls, 4 mL of freshly prepared YE8 solution was labeled as tube D; and 20 μL seeds of YE8, YEHK21, and YEHK21-YE8 in 3.8 mL of deionized water were labeled as tubes E, F, and G, respectively. The samples were incubated for 12 days at pH 6.5, a condition in which YE8 remains soluble and unfolded. The experiments were done in triplicates and the average taken. Fluorescence Spectroscopy. Fluorescence spectra were measured in a 1 cm rectangular quartz cell using a Jobin Yvon Fluoromax-3 spectrofluorometer (Jobin Yvon, Edison, NJ). On excitation at 450 nm the emission spectrum from 460 to 500 nm was recorded. Initially, the fluorescence of 2 mL of ThT solution (20 mM in 100 mM potassium phosphate buffer, pH 7.4) was measured as a background, then 20 μL (complete sample) or 50 μL (supernatant) of the sample solution was added and quickly mixed using a pipet. Fluorescent intensity change at 482 nm was obtained by subtracting the background spectrum from the spectrum of sample-ThT mixture. UV Absorption and Circular Dichroism (CD) Spectroscopy. The far-UV CD spectra of the 44 μM peptide solutions were measured in 1 mm temperature-controlled quartz cell using Jasco J-720 spectrophotometer (JASCO, Tokyo, Japan). The following parameters were utilized: a bandwidth of 1 nm, resolution of 0.5 nm, scan speed of 100 nm/min, and a response time of 0.3 s. Six accumulations were averaged. UV absorption spectra were measured in a 0.05 cm quartz cell using Hewlett-Packard HP 8452 diode array spectrophotometer. Atomic Force Microscopy (AFM) Measurements. For each sample, a 30 μL aliquot was placed on a graphite surface (HOPG) for 5−10 min, and then the samples were rinsed with deionized water. Three-dimensional measurements were collected in air using standard Si cantilevers (AC240TS, Olympus America, Center Valley, PA) and the tapping mode technique of AFM (MFP-3D, Asylum Research, Santa Barbara, CA). Collected images were then analyzed with IGOR Pro 6 (WaveMetrics, Lake Oswego, OR). Resonance Raman Measurements. A 197 nm laser beam (∼1 mW, indigo-S laser system, Coherent, Santa Clara, CA) was used to focused into a spinning NMR tube (5 mm outer diameter, 0.38 mm wall thickness) containing 150 μL solution. A custom-built subtractive double spectrograph equipped with a Roper Scientific Spec − 10:400B CCD camera (liquid-nitrogen cooled) was utilized for recording Raman spectra. GRAMS/AI (7.01) software was used for Raman spectroscopic data processing. Transmission Electron Microscopy (TEM) Measurements. The carbamoylated chimeric sample (YHK21-YE8, 44 μM) was adsorbed to a Formvar-coated slot grid for 5 min, followed by 2 min incubation with 1% uranyl acetate. The grid was then washed twice in distilled water (10 s) and blotted dry. A grid incubated in distilled water prior to uranyl acetate served as a staining control. Samples were observed in a Zeiss 910 TEM at 80 kV. YEHK21 and YEHK21-YE8 Sample Preparation. Each carbamoylated and purified sample was dialyzed against doubly distilled water using a dialysis membrane with 3500 Da cut-off. After dialysis the polypeptides were centrifuged for 20 min at 15000 g and 4 °C. After centrifugation, YEHK21 was separated into two phases: gelatinous and aqueous, which were subsequently separated. The gelatinous phase was resuspended by vortexing with an equal amount of water and then the solution was centrifuged. The supernatant (S1) was then separated and the polypeptide concentration adjusted to 44 μM. The supernatant (S1) was used as the YEHK21 stock solution. YEHK21-YE8 on the other hand formed no discernible insoluble material after dialysis. After centrifugation the concentration of the peptide was adjusted to 44 μM and pH was controlled at 6.5 ± 0.5. Samples (10 mL) of both YEHK21 and YEHK21-YE8 were used as stock solutions for the experiment. After incubation for greater than 4 days at room temperature, both samples could be separated into gelatinous and aqueous phases. At any given time, the aqueous phase was used for analysis.

previous study on charge distribution using YEHK21 and YE8, it was found that the precise distribution of electrostatic interactions within polypeptides, rather than simply the reduction or elimination of these interactions, can promote amyloid fibril formation.41 YEHK21-YE8 was prepared to validate the hypothesis that the YEHK21 domain can induce folding of the YE8 domain and afford insight into the role played by charge on folding and aggregation.

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EXPERIMENTAL SECTION

Materials. Restriction endonucleases BamH1, EcoR1, and BsaI, T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA). A BsaI isozyme, EcoR1, and Rapid DNA Ligation Kit were purchased from Fermentas Inc. (Hanover, MD). Benzonase was purchased from Novagen, Inc. (Madison, WI). Inoue ultracompetent and electrocompetent cells of XL1-Blue (Stratagene, La Jolla, CA) and DH5αF′ (Invitrogen, Carlsbad, CA) were prepared according to standard methods.68 BLR(DE3)pLysSRARE was prepared from BLR(DE3) (Novagen, Inc., Madison, WI) transformed by pLysSRARE isolated from Rosetta(DE3)pLysS (Novagen, Inc., Madison, WI), as previously reported,64,69 and the frozen stock of the chemical competent cells (containing 10% glycerol to prevent autolysis upon thawing) was prepared by standard calcium chloride method and stored at −80 °C.68 Plasmids pUC18 and pET-28a-c were obtained from Bayou Biolabs (Metairie, LA) and Novagen, Inc. (Madison, WI), respectively. Plasmids and DNA fragments separated by agarose gel electrophoresis were purified using QIAprep Spin Miniprep Kit and QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA). Western blotting was done using SuperSignal West HisProbe kit (Pierce/ Thermo Fisher Scientific Inc., Rockford, IL). Large scale purification of polyhistidine-tagged repetitive polypeptides was performed with NiNTA Superflow affinity columns (Qiagen Inc., Valencia, CA). Preparation of Recipient Vectors. pUC18 (10 μg) and pET28a-c (1 μg) were double-digested with BamH1-EcoR1 and NcoIBamH1, respectively, under the recommended conditions and then purified by agarose gel electrophoresis. Excised gels containing the desired fragments were purified using QIAquick Gel Extraction Kit and eluted with TE (pH 8.0, 100 μL) to give pUC18/BamH1-EcoR1 for assembly of coding DNA sequences or eluted with TE (pH 8.0, 40 μL) to give pET-28/NcoI-BamH1 for expression of the coding sequences, respectively. Construction of Expression Vector with Genes Coding High Molecular Weight Polypeptide (YEHK21-YE8). The preparation of the genes coding for YE8 and YEHK7 in pUC18 and H6-YEHK21H6 in pET-28 were reported previously.64,69 YEHK21 in pUC18 was constructed by oligomerization of YEHK7 following previous reports.64,69 E. coli bearing the YE8 or the YEHK21 genes in pUC18 were grown in (Terrific Broth) with ampicillin cultures overnight at 37 °C. The plasmids were harvested and then digested with BsaI. The genes were oligomerized with cloning adaptors69 and the genes with greater than 2000 bp on agarose gel were separated, ligated to pUC18/ BamH1-EcoR1 and transformed into XL-1 Blue. Select colonies on ampicillin plates were investigated for the YEHK21-YE8 sequence by plasmid miniprep and BamH1 digestion. The plasmids harboring the desired length inserts were sequenced. The YEHK21-YE8 containing plasmid was digested by EcoR1. The YEHK21-YE8 DNA fragment was ligated with H6−H6 expression adaptors to form the desired H6YEHK21-YE8-H6 DNA sequence. Following purification by agarose gel electrophoresis, the DNA was ligated with the previously digested recipient expression vector pET28/NcoI-BamH1. The expression vector harboring the H6-YEHK21-YE8-H6 coding sequence was used to transform the BLR(DE3)pLysSRARE expression host. The protocol for expression and purification of the carbamoylated polypeptides is described elsewhere.69 Seeding Experiment. For template studies, 3.98 mL freshly prepared 44 μM YE8 solution was dispensed in three sterile polystyrene plastic tubes (12 OD × 75 mm L, Fisher Scientific Inc., Waltham, MA) labeled A−C. A total of 20 μL of 44 μM fibrillated YE8 solution then sonicated was added to tube A (0.5% templating material 2994


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RESULTS Secondary Structure Using Far-UV CD. Far-UV CD has been extensively used for the characterization of protein secondary structure. Following dialysis, CD measurements of the chimeric construct were recorded daily. The spectra from days one and three are summarized in Figure 2

It is known from previous studies that YE8 forms amorphous aggregates at pH 5.41 YE8 is unfolded and soluble at pH 6.5 with the CD spectrum that of an unordered polypeptide. At pH 3.5 the CD spectrum of YE8 (Figure 2C, green) has a strong positive band at ∼197 nm and a strong negative band at ∼215 nm. The potential to distinguish the constituent contributions to the CD spectrum of a mixture of conformers by subtraction of basis spectra was demonstrated by Martens et al.70 Interestingly, a quantitative subtraction of YEHK21 spectrum from that of YEHK21-YE8 yielded a spectrum (YE8*, YE8 induced)71 that resembled the ∼8 nm red-shifted spectrum of folded YE8 (after ∼40 days incubation at pH 3.5) (Figure 2C). This contribution to the YEHK21-YE8 CD spectrum offers a rationale for the diminished negative and positive maxima intensities. The CD spectra of YEHK21-YE8 and YEHK21 differ from the characteristic CD spectra of globular proteins, with the differences attributed to a strong contribution from either (i) the interaction of tyrosine or histidine side chains to form πstacked arrays (Figure 1) or (ii) a strong contribution to the spectrum from the regularly peptide turn repeats.64 However, Raman spectra of YEHK21-YE8 and YEHK21 (see section below) collected on day three were consistent with the presence of polypeptides with a high β-sheet content. Characterization by TEM. TEM measurements enabled accurate measurement of the fibril width. The TEM micrograph of YEHK21-YE8 stained by 1% uranyl acetate (Figure 3) showed that YEHK21-YE8 formed linear assemblies. A probability density plot generated from measurement of 93 fibrils yielded a predominant fibril width of 7.5 ± 1.0 nm (Figure 4). This width is consistent with a structure made up of 2 β-sheets side-by-side with the hydrophobic groups packed closely together. The width (turn-strand turn), as determined from computed molecular model, is 3.4−3.8 nm (Figure 5) and is consistent with earlier YEHK21 fibril measurements.64 Fibrils can be seen on close examination to overlap on intersection implying fibril formation occurred in solution prior to deposition on the Formvar surface. Characterization by AFM. Tapping mode atomic force microscopy (TM-AFM) was utilized to confirm the structure of YEHK21-YE8 and to investigate the fibril morphology. A notable feature of the topographic image of YEHK21-YE8 deposited on HOPG is the presence of linear, nonbranching fibrillar structures (Figure 6A). The thickness of a typical fibril is shown in Figure 6B,C, where the dimensional regularity of the fibril is clear. A more complete data analysis of multiple fibrils shows the presence of two main fibril populations, one with a thickness of 1.44 ± 0.31 nm and a second smaller and less uniform population with a thickness of 2.87 ± 0.89 nm (Figure 6D). The principal fibril thickness of 1.44 nm, attributable to a single fibril (Figure 6C), is twice the computed thickness of a YEHK21 β-sheet assemblage (Figure 5). YEHK21-YE8, therefore, appears to preferably assemble in a bilayer fibrillar structure similar to that reported for YEHK21.64 The second population of fibrillar structures with a thickness of 2.8 nm is most likely the result of two (or more) fibrils entangling, resulting in determination of a thickness with a broad standard deviation. Comparison of YEHK21-YE8 with YEHK21 and YE8 by DUVRR. Figure 7 shows the DUVRR spectra of YEHK21-YE8 (blue), YEHK21 (green), and YE8 (red) incubated at various length of time and at different conditions. YEHK21-YE8 and

Figure 2. (A) Far-UV CD spectra of YEHK21-YE8 measured on days 1 (red) and 3 (black). (B) Far-UV CD spectra of YEHK21 measured on days 1 (red) and 3 (black). (C) Comparison between YE8* (red) and YE8 (green) spectra. YE8* spectrum was obtained as YEHK21YE8 spectrum minus YEHK21 spectrum.

The CD spectra of both polypeptides (YEHK21-YE8 and YEHK21) consisted of a strong positive band at ∼197 nm, a strong negative band at ∼207 nm and a broad shoulder at ∼220 nm. This spectral feature is the CD signature of the β-sheet in the YEHK family.64 The strong positive band increased for both polypeptides in the first 3 days but did not change after the third day. The gradual increase indicated an increase in βsheet content. It was not surprising that the YEHK21-YE8 CD spectrum was predominantly that of YEHK21 since the YEHK21 domain constitutes 84% of the whole polypeptide (Figure 2A,B). However, the spectra of both polypeptides differ in that YEHK21 spectrum had a deeper negative maximum and a more intense positive maximum compared to that of YEHK21-YE8. 2995


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Figure 3. (A) TEM micrograph of YEHK21-YE8 on Formvar; (B) TEM micrograph of YEHK21.74

YEHK21 were measured on the third day of incubation at pH 6.5. These spectra are compared with that of YE8 incubated for 45 days at pH 3.5. The spectra are dominated by contributions from vibrational signatures from tyrosine and the amide bonds of the polypeptide backbone. The contribution from the polypeptide backbone consisted of amide I, amide II, amide II, and CαH bending modes while the contribution from the tyrosine was comprised of bands at 1175, 1206, 1604, and 1620 cm−1. Characteristic DUVRR signal enhancement of amide bands, especially amide I peak at 1680 cm−1 observed indicated folded YEHK21-YE8 (blue), YEHK21 (green), and YE8 (red) polypeptides (Figure 7). The relative contribution of the tyrosine Raman signature at 1618 cm−1 was greatest in YE8 compared to YEHK21-YE8 and YEHK21, in agreement with primary sequences of these polypeptides. Effects of Various Seeds on YE8 Fibrillation at pH 6.5. The amyloid probe, ThT, was employed to follow fibril development in the seeded YE8 samples. Figure 8 shows the effect of adding various seeds (i.e., preformed YEHK21, YEHK21-YE8, and YE8 fibrils) on a solution of freshly prepared YE8 at pH 6.5 followed by ThT. Unseeded YE8 incubated at pH 6.5 does not form fibrils. The changes in the fluorescence intensity at 482 nm of the YE8 sample incubated with YEHK21-YE8 seeds (sample C) clearly indicate the formation of fibrils, without any lag phase. This rate of folding is in sharp contrast to the 7 day lag associated with folding of unseeded YE8 incubated at the established optimum pH of 3.5.67 YE8 samples seeded with YE8 and YEHK21 (samples A and B, respectively) during the same incubation period of 12 days, remained unfolded and consequently no fibrillation was observed as the fluorescence intensity of these samples did not increase significantly. These results were supported by the CD spectroscopic analysis. The CD spectra of YE8 with YEHK21-YE8 seeds showed characteristic bands for unfolded and β-sheet folded YE8 on days 1 and 12, respectively. Again, CD spectra of YE8 seeded with YE8 or YEHK21 remained unchanged and had a broad negative shoulder at ∼200 nm characteristic for unordered structures for both samples on days

Figure 4. Probability density plot of fibril width from the TEM image of YEHK21-YE8. 93 samplings were used to generate the distribution profile.

Figure 5. YEHK repeat units with γ-turns forming antiparallel β-sheet structure stabilized by H-bonds. Width (turn-strand-turn) of computed model, 3.4−3.8 nm; thickness, 0.72 nm.

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Figure 6. AFM topograph of YEHK21-YE8 fibrils on HOPG and freehand lines drawn along fibrils length for analysis of their height; (B) Magnification of selected fibril (yellow dashed box in panel A). (C) Height measurement along the length of a representative fibril (in panel B). (D) Data distribution (histogram) of fibrils height captured by AFM in panel A, binned in 0.1 nm increments, with the resultant fitting curve (solid blue) derived from combination of multiple Gaussian distributions (dash black). Individual peaks are indicated as mean ± standard deviation.

1 and 12. To ascertain that the fluorescence was not derived from the seed, a control sample of 3.8 mL water, pH 6.5, and 20 μL of fibrillated YEHK21-YE8 was also measured. The ThT signal was negligible (data not shown) effectively eliminating the possibility that the increase in ThT intensity was derived from the seeds alone. The CD spectra of the control sample taken on days 1 and 12 produced no discernible signal.

N-terminus bearing a hexahistidinyl track and the C-terminus seamlessly linked to the N-terminus of YE8. The C-terminus of the YE8 tract is terminated with another hexahistidinyl repeat sequence. The proposed model of the folded construct is shown in Figure 1C. Although the hexahistidinyl sequence was originally introduced to provide a metal binding site, it was subsequently found that the hexahistidinyl tract influences folding and aggregation in a pH-dependent manner. Modulation of charge distribution within the β-sheet forming domain under specific conditions of acidity, polypeptide folding, and coacervation was strongly influenced.41 These same principles and influences can be used to explore the effect of charge distribution on the influence on chimeric peptide folding. The covalent attachment of the protofibrillar assembly (YEHK21 domain) to the disordered sequence enables assessment of the influence of the ordered β-sheet structure on folding of the YE8 domain without the need to dissect the role of intermolecular interactions.

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DISCUSSION Design of Polypeptides. The previously described strategy for the biosynthesis of β-sheet forming repetitive and block copolymerized peptides, based on the pioneering work of Tirrell,72 was used to couple the genes coding YEHK21 and YE8 to form the chimeric construct YEHK21-YE8. Common to both YEHK21 and YE8 are the GAGAGA β-strand repeats. The repeats are terminated by GY, GE, GH, and GK dyads. The YEHK repetitive unit has four β-strand forming GAGAGA repeats separated by GY, GE, GH, and GK known to facilitate β-turns.72 The 62 kDa repetitive chimeric polypeptide, YEHK21-YE8, is comprised of 21 repeats of YEHK with the 2997


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Table 1. Predicted Charges of YEHK21-YE8, YEHK21, and YE8 Polypeptides, the β-Sheet Core, and Different Amino Acids at Different pH 6.5a H6YE8H6 strand

H6YEHK*21H6

H6YEHK*21YE8H6

charge

strand

charge

strand

charge

8E 8Y

−8 0

YE8 2·H6 total

−8 6 −2

21E 21Y 21H 21K*b H6YEHK*21 2·H6 total

−20.9 0 10.5 0 −10.4 6 −4.4

29E 29Y 21H 21K*b YEHK*21YE8 2·H6 total

−28.8 0 10.5 0 −18.3 6 −12.3

a The prediction was based on the pKa values for isolated amino acid residues using Protein Calculator v3.3. bHomocitrulline (K*) was modeled using values for glutamine.

Figure 7. Excited Raman spectra (197 nm) of YE8 (A), YEHK21 (B), and YEHK21-YE8 (C) polypeptides. YE8 spectrum was collected after incubation for 45 days at pH 3.5. YEHK21 and YEHK21-YE8 spectra were collected after a week of incubation at pH 6.5.

Folding of the YEHK21 and YE8 Domains at pH 6.5 in the Chimeric Construct. With 672 amino acid residues, the YEHK21 domain has fast folding dynamics that can be attributed to a less frustrated free energy funnel.64 As mentioned, the folding and aggregation of YEHK21 are strongly influenced by electrostatic interactions.41 The charges borne by YEHK21 and YE8 and the location of those charges were calculated at pH 6.5 (Table 1). The predicted charge distribution within polypeptides is schematically depicted in Figure 9. At neutral pH, strong attractive interactions between

Figure 9. Schematic representation of charge distribution within βsheet of model chimeric polypeptide, H6YEHK21-YE8H6 (C) and intact H6YEHK21H6 (A) and H6YE8H6 (B) polypeptides at pH 6.5 (as it is shown in Table 1). The squares represent the unit containing GAGAGA strand and specific turn; the ovals stand for hexahistidinyl tracks. The red, blue, and white colors represent the positively charged, negatively charged, or neutral amino acids. Charge distribution of YE8 at pH 3.534 (D) is included for comparison.

Figure 8. YE8 templated by seeds of YE8, YEHK21, and YEHK21-YE8. YEHK21-YE8 seeds accelerated the β-sheet formation and fibrillation of YE8 at pH 6.5. 2998


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altered ensemble of conformations required for folding. This observation is consistent with the fact that nonamyloidogenic proteins can neither induce or accelerate amyloidosis,59 as both parent polypeptides (YEHK21 and YE8) can ultimately form amyloid-like fibrils just under different conditions. Transmission Barrier and Seeding of YE8 with YEHK21-YE8. Usually the addition of seeds obtained from amyloid fibrils of a foreign protein does not affect fibrillation kinetics of a native molecule. This phenomenon is explained by the existence of a cross-species or transmission barrier.74,75 The core structure of the amyloid fibrils is a consequence of extended hydrogen bonding between β-strands perpendicular to the fibril axis.76 However, different proteins and peptides have different side chains that influence both the propensity of amyloid formation and the structural details such as the separation between the β-sheets or the way protofilaments assemble to form fibrils.77,78 Despite the presence of the transmission barrier, crossseeding has been shown to occur in a variety of systems.54−56,60,61 From this study, it is apparent that promiscuous seeding is possible but the importance of the nature of the nucleus or seed to the process has been underscored. The YEHK21 seed did not trigger the folding and hence fibrillation of YE8 polypeptide, yet the YEHK21-YE8 seed promoted folding and fibrillation of YE8 at pH 6.5 (Figure 8). YEHK21-YE8 seed was made of two domains, wherein one domain (YE8 domain) bore striking sequence similarity to the seeded peptide (YE8). Apparently the YEHK21-YE8 seed easily captured unfolded YE8 polypeptide and redirected folding at pH 6.5. YE8 seeds, on the other hand, did not accelerate the fibrillation in this unfavorable environment (pH of 6.5). It was earlier recognized that folded YE8 monomer will denature at pH 6.5, but there is no evidence that reduced monomer stability affects the stability of the seeds at pH 6.5. Seed disaggregation may be sufficiently slow that this instability does not effectively reduce seed concentration. The CD of folded/fibrillated YE8 seeded with YEHK21-YE8 (Figure 7) resembles that of folded/fibrillated unseeded YE8 (Figure 2C). Together, these results suggest the following: (i) a YEHK21-YE8 seed triggers the folding of YE8, yet does not change the conformational arrangements of fibrillated YE8, forcing it to fold into the same fibrils which YE8 would otherwise form unseeded at pH 3.5; and (ii) the red-shift in Figure 2C may be a consequence of either YEHK21 or YE8 forming conformationally distinct fibrils. It is nonetheless important to acknowledge that this interpretation assumes that the YEHK21 part of the chimeric protein is not significantly influenced by the presence of the YE8 domain. Further analysis to determine this by dissecting the polypeptide backbone67 is underway in our lab.

the positively charged lysine and negatively charged residues at the edge of native YEHK21 led to the precipitation of amorphous aggregates. In contrast, following carbamoylation of lysine to form neutral homocitrulline, the uncompensated negative charge along the edge of the YEHK21 sheet promotes formation of very regular, amyloid-like fibrils (Figure 9A). Like YEHK21, the folding and aggregation of the YE8 domain is strongly dependent upon electrostatic interactions. Extensive studies of YE8 at pH 3.5 have shown that there is a decrease in the net charge and concomitant decrease in electrostatic repulsion between the glutamic acid moieties facilitating in YE8 aggregation into well-defined amyloid fibrils (Figure 9B).41,65 At pH 6.5, YE8 polypeptide is unfolded largely due to the accumulation of negative charge along a single edge, a result of the ionized glutamic acid residues and the lack of charge on the opposite edge bearing tyrosines at the turns (Figure 9D).41 The charge distribution of the YEHK21-YE8 is a composite of the two domains. The YE8 domain has a high negative and localized charge on one edge yet charges are distributed alternately along the YEHK21 domain (Figure 9C). A difference CD spectrum YE8* has been obtained where the contribution of YEHK21 has been subtracted from the YEHK21-YE8 spectrum. The comparison of the YE8 CD spectrum with the YE8* spectrum (Figure 2C) clearly illustrates that the residual spectrum YE8* can be attributed to the β-sheet folded YE8 domain. The similarity of the TEM and AFM images of YEHK21-YE8 (Figures 3 and 6) with the previously published microscopy of YEHK21 and YE8 was consistent with the participation of both the YE8 and YEHK21 tracts of the chimeric construct being involved in bilayer fibril formations. Molecular Interaction between YEHK21 and YE8 Domains in the Chimeric Construct at pH 6.5. The intramolecular interaction can be related to fibrillation resulting from the intermolecular recruitment of monomer proteins to a protofibrillar assembly. The ability of the folded structure at the point of growth of an amyloid fibril to serve as a template for the recruitment of monomer can be likened to the propensity of domains and subdomains of globular protein clusters to pack with complementarity and high specificity57 in seeding experiments. When the folded and unfolded domains are covalently linked, entropic barriers to interaction of the folded polypeptide−unfolded polypeptide are eliminated, yet the energetics of polypeptide−solvent interactions is largely unchanged. There is evidence of cooperativity at the molecular level of the folded polypeptide-unfolded polypeptide domains in YEHK21-YE8 dialysate. The dialysate did not separate into gelatinous and soluble phases like the YEHK21 polypeptide. Presumably, the unfolded YE8 domain inhibited the formation of the aggregates that result in phase separation. On incubation for as few as 3 days, the folded YEHK21 domain gradually induces the folding of the unfolded YE8 portion even under conditions previously established as unfavorable (pH 6.5) for YE8 folding. The propensity alanylglycyl dyads (AG)3, common to both domains, to form antiparallel strands72 overcomes the electrostatic repulsions of the adjacent ionized glutamic acid residues in the YE8 portion of the molecule. The adjacent strands apparently zip together to form β-sheet through linear hydrogen bonds. Previously we have found that YE8 folds to a β-sheet conformation only after a tertiary structure rearrangement.73 It is therefore highly likely that the presence of the folded YEHK21 domain can enhance the transformation of the unordered YE8 domain to the necessary

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CONCLUSION A chimeric polypeptide, YEHK21-YE8 was designed and constructed de novo. The chimeric polypeptide was found to form well-ordered fibrillar structures at pH 6.5, whereas the YE8 polypeptide alone would otherwise be soluble, unordered, and unfolded. Our results suggest that the YE8 domain in the chimeric polypeptide was induced to fold at pH 6.5 by YEHK21 domain that aggressively folds under the conditions studied. The energy barrier frustrating the folding of YE8 domain was easily overcome by the induction of the folded YEHK21 domain. The lack of this definitive energy barrier in 2999


Biomacromolecules

Article

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amyloid could explain cross seeding and the coexistence of PMDs. Implications to the Deposition of Coexisting Intrinsically Unordered Proteins. Electrostatic interactions strongly influence the folding and aggregation state of the YEHK21 domain in the model chimeric polypeptide. Folding of the YE8 domain, with an uncompensated net charge, was induced by the YEHK21 domain that acted as a foreign seed. From these findings, in vivo, intermolecular interactions may result in local environmental changes that affect similarly the promotion of induced folding and fibrillation of one protein which in turn would act as seed to induce the folding of a second protein that could otherwise require a longer incubation time. Amyloid fibril formation might then be a preferable pathway for intrinsically unfolded proteins that is triggered by seeds having similarity in primary sequence or structural similarity. Cross-seeding between diverse misfolded proteins has become a feasible mechanism.55,57,58,60 This mechanism may be of utmost importance in the understanding of the pathogenesis of neurodegenerative diseases where one of many such diseases would serve as a precursor for another. In vitro, induced folding is usually promoted by global environmental changes, where intermolecular assistance could be a factor. The long lag phase in the folding kinetics of YE8 may be a consequence of the time required for folding sufficient to form protofibrils. Those protofibrils in turn provide the intermolecular interactions necessary to overwhelm the intrinsic barrier toward folding. On fusion to the YEHK21 domain the barrier to YE8 folding was apparently reduced by intramolecular interactions with the surrogate protofibril, the folded YEHK21 portion of the molecule.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Michael Koonce at Wadsworth Center, New York Department of Health, for the TEM measurements. This research was supported by the National Science Foundation (CHE-0809525 and CHE-0957544) to J.W., and the support of the U.S. Department of Energy (DE-FG02-90ER14114 and DE-FG02-50ER46249) and NSF-NIRT (CTS-0304055) to G.B.

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Chimera-Induced Folding: Implications for Amyloidosis

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