Charge Distribution and Amyloid Fibril Formation: Insights from

Biomacromolecules 2010, 11, 1721–1726

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Charge Distribution and Amyloid Fibril Formation: Insights from Genetically Engineered Model Systems Natalya I. Topilina, Vitali Sikirzhytsky, Seiichiro Higashiya, Vladimir V. Ermolenkov, Igor K. Lednev, and John T. Welch* Department of Chemistry, University at Albany, State University of New York, Albany, New York, 12222 Received December 23, 2009; Revised Manuscript Received May 7, 2010

The influence of electrostatic interactions on protein amyloidogenesis has been investigated using de novo designed repetitive polypeptides YEHK21 [GH6[(GA)3GY(GA)3GE(GA)3GY(GA)3GE]21GAH6] and YE8 [GH6[(GA)3GY(GA)3GE]8GAH6]. The β-sheet forming polypeptides were designed with identical β-strands but with variable substitution at the turns that enable precise location of charged residues (Topilina et al. Biopolymers 2007, 86 (4), 261-264; Topilina et al. Biopolymers 2010, submitted for publication; Topilina et al. Biomacromolecules 2006, 7 (4), 1104-11). Solubility, folding, and aggregation of YEHK21 and YE8 were shown to be controlled by charge distribution. Under those conditions favoring the development of charge, YEHK21 and YE8 have significant propensities to form intermolecular β-sheet assemblies illustrating the potential of charged polypeptide chains to form ordered amyloid aggregates even in the absence of additional environmental factors such as the presence of polyelectrolytes, salts, and so on.

Introduction Under some conditions, the coacervation of proteins and peptides to form well-ordered fibrils appears to be an intrinsic property of a polypeptide chain.4 Modern molecular biology and medicine have focused on understanding this process given the prevalence of β-sheet enriched fibrillar assemblies of proteins and peptides that are associated with devastating diseases such as systemic amyloidoisis and neurodegenerative disorders.4-6 The propensity of peptides and proteins to form intermolecular β-sheets and to further aggregate into amyloid-like structures is determined by generic properties such as hydrophobicity, net charge, and the tendency of certain side chains to promote the formation of β-sheet structures.7-9 In particular, electrostatic interactions derived from the number and position of charged amino acids can determine the conformational stability of the polypeptide chain and the tendency of a sequence to form amyloid-like aggregates. It is known that the net charge of a protein is inversely related to the tendency of that material to aggregate;10,11 accordingly, charge neutralization can promote protein aggregation.10,11 However, there are numerous examples of amyloid fibril formation from charged peptides and proteins both in vivo and in vitro.5 Amyloid diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and Creutzfeld-Jacob involve the deposition of natively disordered proteins12,13 that have a significant charge under physiological conditions13-15 and yet in the disease state form amyloid fibrils.12 Many amyloidogenic proteins aggregate in vitro under acidic pH where significant charge can be present.12,16-20 The formation of fibrillar assemblages by charged proteins and peptides results in aggregates with a pronounced polyelectrolyte nature.21 Various electrolytes and polyelectrolytes have been reported to interact with amyloid peptides, proteins, and corresponding aggregates. Electrolytes and polyelectrolytes not only bind amyloid fibrils,22-25 but * To whom correspondence should be addressed. E-mail: jwelch@ uamail.albany.edu.

promote in vitro fibrilogenesis,22,26 affecting the formation of nucleation sites27 and seeds28 as well as fibril stabilization.29,30 We have postulated that the likelihood of a polypeptide chain bearing a significant charge to form amyloid aggregates is determined in large part by the location of the charges within an ordered assemblage such as an amyloid fibril. To test this hypothesis and further probe the influence of electrostatic interactions, two model repetitive polypeptides, GH6[(GA)3GY(GA)3GE]8GAH6 (YE8) and [GH6[(GA)3GY(GA)3GE(GA)3GH(GA)3GK]21GAH6] (YEHK21), designed31 ab initio to generate regular arrays of charged amino acid residues upon folding, have been studied. These polypeptides are proposed to form identical GAGAGA β-strands similar to the β-strand sequence of Bombyx mori silk, (GAGAGS)n, and other de novo designed polypeptides, (GAGAGX)n.32-34 The turns are decorated with specific amino acids to control folding and aggregation.1-3 Both polypeptides form β-sheets1,35 and assemble into amyloid-like aggregates.3 These molecules have previously been utilized to study folding,35 the aggregation of intrinsically unfolded proteins,1 and the influence of strand number and strand length on folding.36 In contrast to these previously published studies that describe the utility of these constructs in the studies of polypeptide folding generally, this work focuses tightly on the influence of the charge state of the polypeptide and on the intramolecular distribution of charge on folding. The comparison of the YEHK system with the YE system will enable dissection of the influence of the backbone histidine and lysine residues on folding.

Experimental Section The protocols for the expression and purification31 of YEHK21 and YE8 can be found elsewhere. Fluorescence, UV Absorption, and CD Spectroscopy. Fluorescence spectra were measured in a 1 cm rectangular quartz cell with a magnetic stirrer using a Jobin Yvon Fluoromax-3 spectrofluorometer. Typically, 275 nm excitation, with 2 nm excitation, 4 nm emission slits, 1 nm data interval, and 0.5 s integration time were used for fluorescence measurements. UV absorption spectra were measured in

10.1021/bm901467a  2010 American Chemical Society Published on Web 06/16/2010

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a 1 mm quartz cell using Hewlett-Packard HP 8452 diode array spectrophotometer. Far-UV CD spectra were measured in 0.05 cm temperature-controlled quartz cell using Jasco J-720 spectropolarimeter. Resonance Raman Measurements. The fourth harmonic (195 nm, ∼2 mW) of the Indigo S laser system (Coherent) is used to generate Raman scattering from a 0.6 mm diameter thermostatically controlled sample solution stream. A custom-built subtractive double spectrograph equipped with a Roper Scientific Spec-10:400B CCD camera (liquid nitrogen cooled) is utilized for recording Raman spectra. The spectral resolution of the system is 4 cm-1. Spectra are analyzed using GRAMS/ AI (7.01) software.1,35 Atomic Force Microscopy (AFM) Measurements. Sample Preparation. After dialysis, aqueous solutions of the polypeptide were used at a pH of 7.0 for further studies. A concentration of 0.12 mg/mL was determined using UV-visible spectroscopy. A total of 50 µL of the aqueous solution was dropped on a 12 × 12 mm sample of highly oriented pyrolytic graphite (HOPG; Veeco Instruments). The polypeptide solution so deposited on HOPG was incubated at room temperature for 30 min and then removed by a micropipet. AFM Imaging Conditions. AFM imaging was performed in resonant tapping mode under ambient conditions with a MultiMode microscope (Digital Instruments) using a Nanoscope IIIa control system. A J scanner was used with a lateral range of ∼125 µm. Images were collected using Nanoscope III software, version 4.42r8, in height and phase mode simultaneously. The standard silicon TESP cantilevers (nominal spring constant, 40 N/m, resonance frequency about 300 kHz, and tip radius less than 10 nm, purchased from BudgetSensors) were used for imaging. The force was minimized during the imaging by choosing a set point corresponding to more than 90% of the free oscillation amplitude of about 12 nm. Typical scan frequency was 1 Hz and images were collected at a resolution of 512 × 512 points. The tip convolution of about 8 nm for features 1.4 nm in height was calculated by imaging the 1.4 nm diameter gold nanoparticles generally used for TEM calibration. Offline Nanoscope image analysis software (version 5.12r3) was used for image analysis. The images were flattened and no further image processing was performed. Transmission Electron Microscopy (TEM) Measurements. Specimens were prepared for transmission electron microscope observation by placing a droplet of the peptide-containing solution on a standard carbon-coated support grid. After 30 min, the solution was removed and the specimen was then stained using uranyl acetate to accentuate the morphology of the structures. Bright field TEM images were recorded on film in a JEOL 200CX operating at 80 kV.

Results YEHK21* Sample Preparation. After expression and purification, YEHK21 was carbamylated by boiling in 8 M urea for 4 h.31 Upon dialysis against doubly distilled H2O, the carbamylated YEHK21 (YEHK21*) exists in a gelatinous phase composed of folded polypeptides assembled into amyloid-like fibrils.3,35 To study the folding and aggregation of YEHK21* at varying conditions, an 8 M urea solution of unfolded polypeptide (solution after polypeptide purification) was dialyzed at 4 °C against aqueous acid or base at pH 10.0, 6.5, 5.0, 3.5, and 2.0. YEHK21* Folding. Upon dialysis, samples were characterized by deep UV resonance Raman spectroscopy (Figure 1). Contributions from vibrational signatures of tyrosine (Y) and the amide bonds of the polypeptide backbone dominate the spectra. The narrow and intense amide I band indicative of a β-sheet37,38 suggests that the YEHK21* existed in a β-sheet conformation at pH 6.5 and was unfolded at pH 2 and 10. Intermediate states with β-sheet and random coil components are observed at pH 3.5 and 5 (Figure 1, blue spectra). Importantly, all folded polypeptides were identified within the gelatinous phase obtained following centrifugation and super-

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Figure 1. Excited Raman spectra (197 nm) of YEHK21* dialyzed against water at pH 2.0, 3.5, 5.0, 6.5, and 10 (blue), and spectra of gelatinous fractions obtained by sample centrifugation and supernatant decantation (red). Table 1. Secondary Structure Composition of YEHK21* at Different pH Valuesa pH

phase

% β-sheet

% random coil

2.0 3.5 3.5 5.0 5.0 6.5 6.5 10.0

initial solution initial solution gelatinous phase initial solution gelatinous phase initial solution gelatinous phase initial solution

0 10 62 76 91 96 97 4

∼100 90 38 24 9 4 3 96

a Contribution of the β-sheet and random coil secondary structure motifs calculated by alternating least-squares approach (two-component decomposition).

natant decantation (Figure 1, red spectra). All supernatants contain YEHK21* in unfolded state (spectra not shown). A more detailed analysis of the Raman spectra of YEHK21* has been reported.3,35 In Table 1, the pH dependence of the relative contribution of β-sheet to the overall secondary structure has been clearly established by careful analysis of the spectral data from Figure 1. AFM Characterization. The morphology of the β-sheet selfassemblages from YEHK21* deposited from solutions (pH 6.5 and 5) on highly oriented pyrolytic graphite (HOPG) was studied by tapping-mode atomic force microscopy (TM-AFM). The topographic investigation revealed the formation of nonbranching amyloid-like fibrils (Figure 2) consistent with previous observations of the polypeptides.1,3,36 Preparation of the YE8 Sample. After expression and purification, a solution containing the YE8 polypeptide was

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Figure 2. TM-AFM topographs of YEHK21* fibrils at pH 5 (A) and at pH 6.5 (B). Table 2. Folding and Aggregation of YE8 Polypeptide1,2 condition pH pH pH pH pH pH pH pH pH

2 3.5 5 6.5 10a 2, saltb 3.5, salt 5, salt 6.5, salt

conformation

aggregation state

β-sheet β-sheet unfolded unfolded unfolded unfolded unfolded unfolded unfolded

amyloid fibrils amyloid fibrils amorphous agg. soluble soluble amorphous agg. amorphous agg. amorphous agg. amorphous agg.

a Spectrum of YE8 at pH 10 is not shown in ref 1. b The effect of ionic strength on the YE8 folding was tested by the incubation of YE8 at different pH the in presence of 0.15 M sodium perchlorate.

Figure 3. Excited Raman spectra (197 nm) of YEHK21* incubated in solution with pH 2 (red) and its gelatinous (blue) and aqueous (green) fractions.

dialyzed against doubly distilled H2O at 4 °C. The repetitive polypeptide remains unfolded under these conditions as determined by Raman and CD spectroscopy.1 YE8 Folding and Aggregation. Folding and aggregation of the YE8 polypeptide1,2 at different pH values are summarized in Table 2. There are likely other contributions to the folding and aggregation state of YE8, however, only the principal or dominant contributor to the conformation and aggregation states are compared. Folding and Aggregation of YEHK21* and YE8 in Charged States. Samples containing YEHK21* at pH 2 and YE8 at pH 6.5 had no detectible β-sheet structure and did not form a gelatinous phase immediately on dialysis. After incubation of YEHK21* for 3 months at room temperature and incubation of YE8 for 1 month at room temperature, Raman spectroscopy revealed low concentrations of the polypeptides with a β-sheet conformation (Figures 3 and 4, blue spectra). On centrifugation, both samples formed gelatinous and aqueous polypeptide fractions that were separately analyzed. The predominance of the β-sheet conformation was clear from the narrow and intense amide I bands in the samples containing aggregated fractions of YEHK21* and YE8 (Figures 3 and 4, blue spectra). The spectra of supernatant solutions are characteristic of disordered polypeptides (Figures 3 and 4, green spectra).

Figure 4. Excited Raman spectra (197 nm) of YE8 incubated in solution with pH 6.5 (red) and its gelatinous (blue) and aqueous (green) fractions.

Figure 5. TM-AFM topographs of YEHK21* fibrils at pH 2 (A) and YE8 at pH 6.5 (B).

Figure 6. Proposed structural models of the repetitive YEHK and YE units. A β-sheet is formed by Ala and Gly residues (only backbone is depicted for clarity). The turns are functionalized by Tyr, Glu, His, and Lys residues forming arrays at the edges of β-sheets.

The AFM studies of the morphology of the polypeptide aggregates in the gelatinous phase revealed the formation of well-defined amyloid-like fibrils (Figure 5).

Discussion YEHK21 and YE8 Polypeptides. The YEHK repetitive unit (Figure 6) has four GAGAGA β-strands separated by the GY, GE, GH, and GK dyads, which are known to facilitate β-turn formation.39 The 51 kDa repetitive polypeptide YEHK21 is comprised of 21 repetitive units of the YEHK monomer, [GH6[(GA)3GY(GA)3GE(GA)3GH(GA)3GK]21GAH6], where both N- and C-termini bear hexahistidinyl tracks. According to the proposed model,33 upon folding, the amino acids at the turn positions differentiate the edges of the β-sheet structure, creating an array of tyrosine (Y) and histidine (H) residues on one edge and an array of glutamic acid (E) and lysine (K) residues on the other (Figure 6). Analogously, the 11 kDa YE8 polypeptide has eight repeats of the YE block, [GH6[(GA)3GY(GA)3GE]8GAH6], with glutamic acid (E) and tyrosine (Y) residues in turn positions (Figure 6). Repetitive Polypeptides as Models for Unfolded Amyloidogenic Proteins. Good models for large intrinsically unfolded amyloidogenic proteins should (i) have simple generic sequences, (ii) not fold as monomers, but fold with intermo-

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Table 3. Predicted Charges of YEHK21* and YE8 at Different pH Valuesa pH

H6YE8H6

YE8

8E

8Y

2H6

H6YEHK*21H6b

YEHK21*b

21E

21K*

21H

21Y

2 3.5 5 6.5 10

12.9 11.4 5.2 -2 -12

0 -0.9 -6.4 -8 -12

0 -0.9 -6.4 -8 -8

0 0 0 0 -4

12 12 11.6 6 0

32.9 30.6 15.2 -4.4 -31.5

20.9 18.6 3.6 -10.4 -31.5

-0.1 -2.4 -16.8 -20.9 -21

0 0 0 0 0

21 21 20.4 10.5 0

0 0 0 0 -10.5

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

lecular assistance to form β-sheets, (iii) aggregate into amyloidlike fibrils, (iv) have precisely positioned charged residues, and (v) develop extremely high charges in response to pH changes. YEHK21 and YE8 are flexible and soluble enough to prevent adventitious monomolecular folding yet fold with intermolecular assistance into β-sheets1,35 and subsequently aggregate into amyloid-like fibrils.1,3 The folding and aggregation of YEHK21 and YE8 have been shown to be strongly dependent on electrostatic forces. At neutral pH, strong attractive interactions between the positively charged lysine (K) and negatively charged glutamic acid (E) residues at the edge of native YEHK21 (Figure 6) led to the precipitation of amorphous aggregates.3,35 In contrast, following carbamylation of lysine to form neutral homocitrulline,31 the uncompensated negative charge along the edge of the YEHK21* β-sheet promotes formation of very regular, amyloid-like fibrils.3 With YE8, at neutral pH, the significantly charged glutamic acid residues (E) of the polypeptide sustain the persistence of the unfolded monomer, while at low pH negative charge reduction facilitates folding and amyloid fibril formation. Charge Distribution within YEHK21 and YE8. Before post-translational modification, the edges of YEHK21 are functionalized by charged lysine (K), histidine (H), and glutamic (E) acid residues (Figure 6). Conversion of the lysine (K) residues of YEHK21 into neutral homocitrulenes to form YEHK21* reduces the number of charged amino acids in the peptide.31 In a further simplification, the repetitive domain of YE8 polypeptide has only one type of charged residue: negatively charged glutamic acid (E; Figure 6). Both YEHK21* and YE8 have two functionally and sequentially different domains: the repetitive tracks that constitute the β-sheet core of an amyloid fibril and the terminal H6 domains that are outside the fibril core.3 The charges of YEHK21* and YE8 and the location of those charges were calculated at various pH values (Table 3). The predicted charge distribution within polypeptides is schematically depicted in Figure 7. At low pH, both polypeptides have high positive charge, while with an increase in pH, the positive charge uniformly diminishes. The effect is a result of both a reduction in the number of positively charged amino acids and increasing charge compensation by negatively charged residues. Near neutral pH, the polypeptides become negatively charged and at pH 10, uniformly negatively charged. For YEHK21* and YE8, charge distribution on the edges of the β-sheet and the terminal unfolded domains of polypeptides have differing dependence upon pH. The peptides may exhibit a combination of positive and negative charges or be uniformly negative or positive. Importantly, the location of charged residues may be restricted to the repetitive β-sheet forming tracks (Figure 7, 5 and 11) and the terminal sequences (Figure 7, 7) or may be distributed across both domains Figure 7, 1). Electrostatic Interactions and Polypeptide Folding and Aggregation. β-Sheet Formation and Charge Distribution. The efficacy of electrostatic control of polypeptide folding and

Figure 7. Schematic representation of charge distribution within β-sheet of modified and intact H6YEHK21H6 and H6YE8H6 polypeptides at different pH (as it is shown in Table 3). The square illustrates 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 residues.

coacervation is determined by charge distribution within the β-sheet forming domains. In accord with previously reported studies,10,40 a decrease in the net charge and concomitant decrease in electrostatic repulsion within the β-sheet forming domain of YE8 results in aggregation into well-defined fibrils (Figure 7, 7 and 8). It was also observed that reduction of local electrostatic repulsion alone rather than a decrease in net charge can enhance amyloidogenic character. For example, at pH 6.5, YEHK21* (Figure 7, 4) has significantly higher charge on the β-sheet edge than can be found with YE8 under the same conditions (Figure 7, 10), yet YEHK21* readily aggregates under these conditions.3 Presumably, aggregation is possible as a result of localization of negative charges at every other turn in YEHK21* in comparison with the location of charges at adjacent turns in YE8. We hypothesize that a precise arrangement of electrostatic interactions within polypeptides, rather than simply the reduction or elimination of these interactions, can promote amyloid fibril formation. The distribution of charged amino acid residues in the polypeptide chain and the ratio of polypeptide charge to molecular weight rather than solely the polypeptide net charge must be considered. Even though YEHK21 and YEHK21* have a very similar sequence, structure, and absolute values for the net charges in the repetitive domains, these molecules have very different properties. These differences are directly related to both the number of charged residues and to the location of those residues within polypeptides. YEHK21 has a greater number of charged amino acids and, importantly, residues lying in close proximity to one and another that were introduced to promote salt bridge formation. The strong attractive electrostatic forces resulting from this charge distribution lead to the rapid collapse of the YEHK21 into amorphous

Charge Distribution and Amyloid Fibril Formation

aggregates. Carbamylation of YEHK21 eliminates the potential for salt bridge formation and leads to local uncompensated charges. The concurrent minimization of strong local attractive interactions with an increase in local electrostatic repulsions in YEHK21* improves polypeptide solubility. Rapid aggregation is suppressed, enabling weak, ordered stabilizing interactions to drive formation of ordered amyloid fibrils. In several proteins, uncompensated charge has been shown to facilitate the formation of ordered amyloid-like assemblages, while screening or compensation of charge can lead to rapid collapse into amorphous aggregates.35,41 This fact is also illustrated by the formation of disordered aggregates on incubation of both YEHK21* (Figure 7, 4)35 and YE8 (Figure 7 7-10)1,2 in the presence of electrolytes. Unfolded Domains. Charge distribution within unfolded domains does not influence polypeptide behavior. YE8 aggregation studies revealed that significant (Figure 7, 9) or even fully uncompensated (Figure 7, 8) positive charge on the terminal domains did not affect the ease of fibril formation. Electrostatic Interactions between β-Sheet Forming and Unfolded Domains. Strong attraction between charged β-sheet forming and unfolded domains in YE8 (Figure 7, 9) leads to aggregation and precipitation. On the contrary, limited attractive electrostatic interactions between β-sheet forming domain and the unfolded terminal H6 tract facilitate polypeptide aggregation. Consistent with electrostatic assistance, at pH 3.5, YE8 coacervates (Figure 7, 8) significantly faster than at pH 2 (Figure 7, 7).2 However, if the net charge on the β-sheet forming domain is too high, electrostatic interactions with unfolded domains cannot compensate for the repulsive effects and the polypeptide remains unfolded and monomeric (Figure 7, 10). Aggregation of Charged Polypeptide Chains. Amyloid fibril formation by charged polypeptide chains was observed in both model constructs. Under some conditions, YE8 (Figure 7, 10) and YEHK21* (Figure 7, 1) have very significant charges in the β-sheet forming amino acid tracts (Table 3). Nonetheless, both polypeptides slowly fold to form β-sheets and, subsequently, fibrils. Consistent with the proposition that charged polypeptide chains can fold and form ordered structures, this observation also describes the capacity of charged polypeptide chains to form fibrils with a cross-β core even under neutral conditions and in the absence of electrolytes. Implications to the Deposition of Intrinsically Unfolded Proteins. Electrostatic interactions strongly influence the folding and aggregation state of the model polypeptides. Importantly, irrespective of the molecular weight, intermolecular assistance to polypeptide folding apparently was observed with both YEHK21*35 and YE8. Moreover, YE8 folding was completely aggregation driven.1,2 Intermolecular assistance in model polypeptide folding is especially important in establishing the relevance of the model to an understanding of the deposition of intrinsically disordered proteins. Proposed mechanisms for intrinsically disordered protein deposition12 require partial refolding as an initial step. In vitro, induced folding is usually promoted by the global environmental changes such as variations in pH, ionic strength, or temperature.12,14 In vivo, intermolecular interactions may result in local environmental changes that affect similarly the promotion of induced folding and fibrillation.12 Electrostatic interactions of intrinsically unfolded proteins with significant charge may also play the major role in local protein destabilization.

Conclusions Two closely related β-sheet forming polypeptides, models of amyloidogenic proteins, were utilized in an investigation of


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the role of electrostatic interactions on high molecular weight polypeptide behavior. For first time the influence of environmental conditions on the folding and aggregation of YEHK21* is described. These results and previously reported characterization of YE8 were analyzed to determine the influence of charge distribution within polypeptide on β-sheet formation and fibrillar aggregation. In particular, the design of the model peptides facilitates the understanding of the effect of electrostatic interactions on β-sheet folding and unfolded domains. Intraand intermolecular electrostatic interactions control folding and also determine the aggregation state of the polypeptides, that is, as monomers or as amorphous or amyloid-like aggregates. The use of peptides with precisely located charges enables the investigation of the importance of electrostatic interactions within the β-sheet forming and unfolded portions of the polypeptides. Importantly, the charged chains of the model polypeptides could undergo intermolecular β-sheet formation and subsequent aggregation into amyloid-like fibrils without the addition of adjuvants such as polyelectrolytes, salts, and so on. Acknowledgment. This material is based on work supported by the National Science Foundation under CHE-0809525.

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