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J. Biol. Chem., Vol. 280, Issue 41, 35069-35076, October 14, 2005

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Sequence Determinants of Enhanced Amyloidogenicity of Alzheimer A42 Peptide Relative to A40^*

From the Department of Chemistry, Princeton University, Princeton, New Jersey 08544

Received for publication, May 26, 2005 , and in revised form, August 2, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Aggregation of proteins into insoluble deposits is associated with a variety of human diseases. In Alzheimer disease, the aggregation of amyloid (A) peptides is believed to play a key role in pathogenesis. Although the 40-mer (A40) is produced in vivo at higher levels than the 42-mer (A42), senile plaque in diseased brains is composed primarily of A42. Likewise, in vitro, A42 forms fibrils more rapidly than A40. The enhanced amyloidogenicity of A42 could be due simply to its greater length. Alternatively, specific properties of residues Ile⁴¹ and Ala⁴² might favor aggregation. To distinguish between these two possibilities, we constructed a library of sequences in which residues 41 and 42 were randomized. The aggregation behavior of the resulting sequences was assessed using a high throughput screen, based on the finding that fusions of A42 to green fluorescence protein (GFP) prevent the folding and fluorescence of GFP, whereas mutations in A42 that disrupt aggregation produce green fluorescent fusions. Correlations between the sequences of A42 mutants and the fluorescence of A42-GFP fusions in vivo were confirmed in vitro through biophysical studies of synthetic 42-residue peptides. The data reveal a strong correlation between aggregation propensity and the hydrophobicity and -sheet propensities of residues at positions 41 and 42. Moreover, several mutants containing hydrophilic residues and/or -sheet breakers at positions 41 and/or 42 were less prone to aggregate than A40 wherein these two residues are deleted entirely. Thus, properties of the side chains at positions 41 and 42, rather than length per se, cause A42 to aggregate more readily than A40.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The deposition of insoluble proteins into amyloid plaque is associated with various diseases including Alzheimer disease, prion encephalopathies, Parkinson disease, and Huntington disease (1-4). In Alzheimer disease, amyloid (A)² peptides aggregate into fibrils, which accumulate as insoluble neuritic plaques. A variety of genetic, neuropathological, and biochemical studies suggests that either the fibrils themselves or precursors on the pathway toward these fibrils play a causative role in Alzheimer disease (1, 5-11).

A peptides are produced in vivo by proteolytic cleavage of the amyloid precursor protein by and secretases (5). Because of inhomogeneous cleavage by secretase, A peptides range in length from 39 to 43 residues. Among these peptides, the 40-mer (A40) and the 42-mer (A42) are abundant in diseased brains (12-14), and these two peptides are the main components of the neuritic plaques in the parenchyma of diseased brains (5, 14).

With the exception of the C-terminal amino acids, Ile⁴¹ and Ala⁴², the sequences of A40 and A42 are identical. Despite their 95% sequence identity, A40 and A42 display dramatically different behaviors both in vivo and in vitro. Biochemical and immunocytochemical studies show that although A40 is major component in cerebrospinal fluid and plasma, senile amyloid plaques formed in vivo are composed primarily of A42 (5, 14-16). Moreover, although both peptides aggregate into fibrils in vitro, A42 does so more rapidly than A40. Concentrated solutions of A40 are stable for days, whereas comparable solutions of A42 aggregate almost immediately (17, 18).

Not only is A42 more prone to aggregate than A40, but the pathways toward aggregation are also different. Recently, Teplow and coworkers (19, 20) found that carefully prepared aggregate-free A40 occurs as monomers, dimers, trimers, and tetramers in rapid equilibrium. In contrast, A42 preferentially forms pentamer/hexamer units (paranuclei) that assembled further into beaded superstructures similar to early protofibrils.

The different aggregation behaviors of A40 and A42 led us to question the role of the two C-terminal residues, Ile⁴¹ and Ala⁴². Is it simply the increased length of A42 that causes its increased amyloidogenicity? Or are particular features of the Ile⁴¹ and Ala⁴² side chains important for amyloid formation? To address these questions, we performed random mutagenesis on positions 41 and 42. The change in aggregation behavior resulting from the mutations was monitored by using fusions of the A42 variants to GFP. Fluorescence of these fusions is inversely correlated with the propensity of the fused A mutant sequence to aggregate (21); GFP fused to wild type A42 does not fluorescence, whereas fusions to less aggregating mutants display increased fluorescence. By correlating the observed levels of fluorescence with the identities of the side chains at positions 41 and 42, we determined the side chain properties at these positions that are responsible for the enhanced aggregation of A42 relative to A40.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction of Libraries of A42-GFP Fusions—Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA), and restriction enzymes were purchased from New England Biolabs. Mutations were incorporated into a synthetic A42 gene (21) by PCR using DeepVent polymerase (New England Biolabs) and an Ericomp Easycycler^TM Twinblock thermocycler. After PCR, the mutagenized A42 gene inserts and the pET 28 vector containing the GFP gene (21) were doubly digested with BamHI and NdeI. The digested insert and vector were then ligated together using T4 ligase. Plasmids were transformed into the XL1-Blue strain of Escherichia coli (Stratagene) and plated for overnight growth on plates containing 50 µg/ml kanamycin.

The primer for random mutagenesis at position 41 and 42 had the following sequence: 5'-TCTTCTGGATCCNNNNNNCACCACGCCGCCCACCAT-3'. The primer for mutagenesis encoding a combinatorial mix of polar residues used the degenerate codon NAN. For a combinatorial mix of hydrophobic residues we used the degenerate codon NTN (where N denotes a mixture of A, G, C, and T).

Screening of Green/White Phenotype—DNA libraries were isolated from E. coli strain XL1-Blue, transformed into BL21(DE3) (23), and plated onto nitrocellulose paper (Millipore NC-HATF 83 mm) on LB plates containing 50 µg/ml kanamycin. Following overnight growth at 37 °C, the nitrocellulose papers were transferred to LB plates containing 50 µg/ml kanamycin and 1 mM isopropyl 1-thio--D-galactopyranoside and incubated at 30 °C for 4 h to induce the expression of the A-GFP fusion protein. Colonies were counted and the green/white phenotype was recorded (21).

Fluorescence Measurements—To enable measurement of the fluorescence of the A42-GFP fusions in vivo, colonies were picked, and cultures were grown in LB liquid media containing 50 µg/ml kanamycin. When cultures reached an absorbance at 600 nm of 0.8, expression was induced by addition of isopropyl 1-thio--D-galactopyranoside to a concentration of 1 mM, and growth was continued for an additional 5 h 30 min at 30 °C. After induction, cultures were diluted in Tris-buffered saline to an A_{600 nm} of 0.15. Fluorescence was measured using a 50B spectrofluorimeter (PerkinElmer Life Sciences) with excitation at 490 nm and emission at 510 nm. Expression of A42-GFP fusions was assessed by removing 200 µl of cell culture and analyzing the whole cell content by SDS-PAGE.

Correlation of Fluorescence with Biophysical Properties—The fluorescence of the A42-GFP fusions in vivo was plotted against the sum of hydrophobicities or against the sum of the -sheet propensities at positions 41 and 42. Hydrophobicities were based on the scale of Kyte and Doolittle (24), and -sheet propensities were based on the scale of Minor and Kim (25). In a further analysis, fluorescence was plotted against the predicted aggregation rates using Equation 1 as developed by Chiti et al. (26).

(Eq. 1)

In Equation 1, _mut and _wt are the aggregation rates of the mutant and wild type sequences, respectively; Hydr is the difference in hydrophobicity, and (G_coil- +G_-coil) is the difference in the propensity to convert from -helix to -sheet. (Note, the two s are added rather than subtracted. This is because subtraction is already accomplished by the definition of the terms. In the first term is subtracted from coil, and in the second term coil is subtracted from .) Charge is the difference in charge, and A, B, and C are empirically determined constants.

Synthetic 42-Residue Peptides—Crude peptides were purchased from the Keck Institute, Yale University, and were then purified using reverse phase HPLC. Their identities were confirmed by mass spectrometry, and purity was assessed by analytical reverse phase HPLC. The purities of the peptides were greater than 92%. After purification, peptides were lyophilized and dissolved in hexafluoroisopropanol (HFIP). HFIP was removed by blowing argon over the sample. Samples were then dissolved in 100 µl of Me₂SO/500 µl of 4 M NaOH. Concentrations were determined using the extinction coefficient of tyrosine.

Aggregation of Mutant Peptides in Vitro—Peptides were dissolved at a concentration of 10 µM in 50 mM NaH₂PO₄, 100 mM NaCl, 0.02% NaN₃ (pH 7.3-7.4). Following incubation at 30 °C for 1 day, samples were centrifuged for 30 min at 60,000 x g. Half of the supernatant was removed and loaded onto reverse phase HPLC to quantify the monomer remaining in solution. To correlate peak size with concentration, a series of standards was run using a range of concentrations of the Asp⁴¹-Gln⁴² mutant. The relationship between peak size and peptide concentration was confirmed using same concentrations of wild type A42, the hydrophobic mutant Leu⁴¹-Leu⁴², and the green control mutant Ser¹⁹-Pro³⁴.

Thioflavin T Assay—Because the kinetics of fibril formation can be affected by small quantities of seeds, preexisting seeds were removed by the following treatment (27). Lyophilized peptides were dissolved in trifluoroacetic acid at 1 mg/ml and sonicated for 15 min. Trifluoroacetic acid was then removed by blowing argon over the sample. The dry sample was resuspended in 2 ml of HFIP. An aliquot corresponding to 0.5 mg of peptide was dried under argon and then dissolved in 300 µl of Me₂SO and mixed with 5 ml of 8 mM NaOH. The solution was then centrifuged at 40,000 x g for 30 min to remove any insoluble material. The supernatant was removed, and phosphate-buffered saline was added to a final concentration of 50 mM NaH₂PO₄, 100 mM NaCl, 0.02% NaN₃. The pH was adjusted to 7.4-7.5 with 200 mM formic acid. 500-µl aliquots were removed every 30 min and mixed with 2.4 ml of a ThT solution (7 µM thioflavin T, 50 mM glycine-NaOH (pH 8.5)). Fluorescence was measured with excitation at 450 nm, and emission at 490 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A High Throughput Screen for A Aggregation—Random mutagenesis of position 41 and 42 can produce 400 possible sequences (including wild type). Synthesis, purification, and characterization of the aggregation behavior of all 400 peptides would be laborious and extremely expensive, particularly because the A42 peptide is notoriously difficult to synthesize and purify (28, 29). Therefore, we developed a high throughput screen using GFP as a reporter tag (21).

The folding of GFP and the formation of its active chromaphore occur relatively slowly (30). Therefore, peptides or proteins fused to the N terminus of GFP can have a dramatic impact on fluorescence. Sequences that aggregate rapidly prevent formation of a correctly folded fluorescent GFP. In contrast, sequences that are soluble or aggregate slowly allow GFP to fold into its native fluorescent structure. In a systematic study using 20 different test proteins, Waldo et al. (31) demonstrated that the fluorescence of E. coli cells expressing fusions to the N terminus of GFP correlated with the solubility of the test protein expressed alone.

We have shown previously that the fluorescence of A42-GFP fusions can be used as an unbiased screen for the sequence determinants of A amyloidogenesis (21). Colonies of E. coli expressing fusions to wild type A42 or to mutants of A42 that favor rapid aggregation are white. In contrast, colonies expressing fusions to soluble (or slowly aggregating) mutants of A42 are green.

Here we used A42-GFP fusions to monitor the phenotypic variation resulting from amino acid substitutions at positions 41 and 42 of A42. This enabled a high throughput screen in which more than 2500 colonies could be screened per plate.

To ensure that the screen had sufficient dynamic range to distinguish a range of phenotypes, we first performed pilot experiments using fusions of GFP to the wild type sequences of either A42 or A40. (The latter construct can be considered as the null mutant in which residues 41 and 42 are not merely substituted but deleted entirely.) At 37 °C both fusions produced white colonies. Thus, at this temperature, the screen does not have sufficient dynamic range. In contrast, at 30 °C, colonies expressing fusions to A42 were white, whereas those expressing fusions to A40 were green. Thus, at this lower temperature, where expression and subsequent aggregation are slower, the difference in aggregation propensity between A40 and A42 is easily distinguished. Further pilot experiments showed that at 37 °C, virtually all mutations at positions 41 and 42 produced white colonies, whereas at 30 °C libraries of mutants at 41 and 42 produced both green and white colonies. Because expression at 30 °C provided excellent dynamic range, all further experiments were performed at this temperature.

A Random Library of Mutants at Positions 41 and 42—A library of random mutants at positions 41 and 42 of A42 was constructed using synthetic oligonucleotides to incorporate random bases (NNN) at the final 2 codons of a synthetic gene encoding A42 (see "Materials and Methods"). The library of mutant genes of A42 was then fused to the 5' end of a gene encoding GFP. In this construct, the A42 sequence is separated from GFP by a 12-residue linker encoding the sequence Gly-Ser-Ala-Gly-Ser-Ala-Ala-Gly-Ser-Gly-Glu-Phe (31).

The library of fusions was then transformed into XL1 Blue cells. Transformation yielded more than 5000 colonies. Because there are only 400 possible combinations of amino acids positions 41 and 42, this library is adequate to sample all (or nearly all) of the possible sequences (TABLE ONE). The library was then extracted from XL1 Blue cells and transformed into BL21(DE3) cells for protein expression and high throughput screening.

View this table: [in this window] [in a new window]   TABLE TWO Green/white phenotypic screening of colonies expressing A(42)-GFP fusions Colonies transformed into BL21(DE3) with plasmids encoding A42-GFP fusions were screened for fluorescence after 4 h of induction at 30 °C. When positions 41 and 42 were both randomized, 91% of colonies were green fluorescent, indicating that many mutations disrupted aggregation. When only hydrophobic residues were allowed at positions 41 and 42, all colonies were white, indicating that random hydrophobic residues support aggregation. In contrast, when only hydrophilic residues were allowed at positions 41 and 42, 87% of the colonies (4160 colonies) were green. The remaining 13% (640 colonies) were white because they did not express the fusion protein. Thus, random hydrophilic residues disrupt aggregation. The other libraries are described in the text.

To confirm this correlation, we produced two additional libraries of mutants. In one library, residues 41 and 42 were mutated to a combinatorial mixture of hydrophobic residues. In the other library, these residues were mutated to a combinatorial mixture of hydrophilic residues. The hydrophobic library was constructed using the degenerate DNA codon NTN to encode a mixture of nonpolar residues including Ile, Leu, Val, Met, and Phe (32). The hydrophilic library was constructed using the NAN codon to encode a mixture of polar residues including Glu, Gln, Asp, Asn, Lys, His, and Tyr (where N denotes a mixture of the four DNA bases).

Both libraries were plated, and protein was expressed at 30 °C. For the 41/42-hydrophobic library, >5000 colonies were analyzed and none were green. Thus, randomly chosen hydrophobic residues at positions 41 and 42 support A42 aggregation.

In contrast, for the 41/42-hydrophilic library, 4800 colonies were analyzed, and 87% of them (4160 colonies) were green. This result indicates that randomly chosen polar residues at positions 41 and 42 disrupt the aggregation of A42.

From the hydrophilic library, only 640 colonies (13%) were white. We tested 64 of these colonies for expression of the A42-GFP fusions, and we found that all but one of them did not express the fusion protein. (Because we used the NAN degenerate codon at positions 41 and 42, it was expected that the library would contain stop codons, which prevent protein expression.) The other white colony expressed normal levels of the fusion protein. Sequence analysis of this clone revealed that it had arginine at both positions 41 and 42. Thus, although most randomly chosen polar residues at positions 41 and 42 interfere with A aggregation, arginine is somehow special and allows aggregation (see below).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Fluorescence of A42-GFP fusions. Cells were cultured in liquid media, and protein expression was induced at 30 °C. After induction, cells were diluted in buffer, and fluorescence was measured at 510 nm. A, mutants with hydrophobic residues at positions 41 and 42 show lower fluorescence, consistent with higher levels of aggregation preventing the formation of the GFP chromaphore. B, mutants with -sheet breakers (Pro or Gly) show a high level of fluorescence. The Glu41-Pro42 mutant shows an exceptional level of fluorescence, indicative of a very low propensity to aggregate, resulting from both charged and proline residues.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Aggregation of synthetic 42-residue peptides. Peptides (10 µM) were incubated at 30 °C for 1 day. Samples were then centrifuged to remove precipitated aggregates, and the supernatant was run on reverse phase HPLC. The size of the peak indicates the amount of monomer that remained in solution. The hydrophilic mutant, Asp41-Gln42, and the non-C-terminal green control, Ser19-Pro34, yielded peaks at the elution time expected for monomers. In contrast, wild type A42 and the hydrophobic mutant, Leu41-Leu42, showed almost no soluble peptide.

Effect of the Site of Mutation—To assess which of the two positions affects aggregation more significantly, we constructed two additional libraries. In each of these libraries one of the two C-terminal residues was held constant, whereas the other was randomized. These libraries were called Ile⁴¹-Random⁴² and Random⁴¹-Ala⁴². (The wild type sequence is Ile⁴¹-Ala⁴².) For each library, nearly 200 colonies were analyzed. The Random⁴¹-Ala⁴² library yielded 37 white colonies (19% white), and 153 green colonies. The Ile⁴¹-Random⁴² library yielded 44 white colonies (24% white), and 141 green colonies (TABLE TWO). Because the fraction of white colonies in the two libraries is not significantly different, we conclude that the wild type residues at positions 41 and 42 contribute to aggregation to a similar extent.

Aggregation of Mutant Peptides in Vitro—Waldo et al. (31) and Wurth et al. (21) demonstrated the validity of GFP fusions as a screen for the aggregation behavior of proteins and peptides. In particular, Wurth et al. (21) showed that the fluorescence in vivo of a mutant of A42 fused to GFP correlated well with the solubility/aggregation behavior of the same mutant in the context of the 42-residue synthetic peptide. In the current study, however, because we have focused on residues at the C terminus of A42, it is reasonable to question whether GFP fusions can produce meaningful readouts for perturbations so close to the fusion linker and GFP.

To address this question, we studied four sequences from our library as synthetic 42-residue peptides: Wild type A42, Asp⁴¹-Gln⁴², Leu⁴¹-Leu⁴², and Ser¹⁹-Pro³⁴. The mutants Asp⁴¹-Gln⁴² and Leu⁴¹-Leu⁴² were chosen as representatives of the hydrophilic green mutants and the hydrophobic white mutants, respectively. The Ser¹⁹-Pro³⁴ peptide is a control. This non-C-terminal mutant was isolated previously by Wurth et al. (21), who found the following: (i) as an A42-GFP fusion, Ser¹⁹-Pro³⁴ was the most fluorescent mutant in their collection; and (ii) as a synthetic 42-residue peptide, it was far more soluble and less prone to aggregate than wild type A42.

The extent of aggregation was compared for the four peptides. Peptide samples were prepared at 10 µM concentration in 50 mM NaH₂PO₄, 100 mM NaCl, and 0.02% NaN₃ (pH 7.3-7.4) and were incubated for 1 day at 30 °C. After incubation, samples were centrifuged at 60,000 x g for 30 min to precipitate the aggregated material. The amount of unaggregated peptide in the supernatant was then quantified by reverse phase HPLC (see "Materials and Methods").

As shown in Fig. 2, the hydrophilic mutant, Asp⁴¹-Gln⁴², and the non-C-terminal green control, Ser¹⁹-Pro³⁴, yielded substantial peaks at the elution time expected for monomers of each peptide. Integration of the peak areas indicated that for these two peptides almost the entire sample remained in solution and unaggregated. In contrast, wild type A42 and the hydrophobic mutant, Leu⁴¹-Leu⁴², showed almost no peaks corresponding to monomeric soluble peptide (Fig. 2). These results confirm that the fluorescent phenotype of the GFP fusions in vivo is a reasonable predictor of the aggregation behavior of the isolated 42-residue peptides in vitro, even for mutants at the C terminus of the peptide.

Rate of Amyloidogenesis—Although the experiments described above demonstrate that aggregation of A42 peptides in vitro mimics the phenotypes of the GFP fusions in vivo, it is important (i) to establish that the aggregated states of these peptides are indeed amyloid, and (ii) to compare the rates of amyloid formation for the different peptides.

View larger version (8K): [in this window] [in a new window]   FIGURE 3. Thioflavin T fluorescence of wild type and mutant 42-residue peptides. Synthetic peptides (20 µM) were incubated in quiescent condition (not agitated). After 1 day of incubation samples were mixed with thioflavin T, and fluorescence was measured at 490 nm. Wild type A42 and Leu41-Leu42 showed thioflavin T fluorescence indicative of amyloid structure.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Kinetics of amyloid formation followed by thioflavin T fluorescence. Purified peptides (20 µM) were agitated at 30 °C. Aliquots were removed every 30 min and mixed with thioflavin T, and fluorescence was measured at 490 nm. Wild type A42 and the hydrophobic mutant, Leu41-Leu42 form amyloid from the outset, whereas the hydrophilic mutant, Asp41-Gln42, like A40, aggregates only after a 90-120-min lag. The control peptide, Ser19-Pro34, showed little or no ThT binding even after 5 h.

Next, we followed the rate of fibril formation under conditions that favor aggregation: agitation, rather than quiescent incubation. Time course measurements of ThT binding showed that wild type A42 and the hydrophobic mutant, Leu⁴¹-Leu⁴², began to form amyloid within a few minutes. In contrast, the hydrophilic mutant, Asp⁴¹-Gln⁴², like A40, began to form amyloid only during the 2nd h of incubation (Fig. 4). (The control peptide, Ser¹⁹-Pro³⁴, showed little or no ThT binding even after 5 h.)

Thus, the behavior of the synthetic 42-residue peptides in vitro validates the results observed for the GFP fusions in vivo. Hydrophobic mutants at positions 41 and 42 mimic wild type behavior, whereas hydrophilic mutants retard amyloidogenesis.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Correlation of the fluorescence in vivo of A42-GFP fusions (except Gly and Pro) with biophysical properties of the residues at positions 41 and 42. A, correlation with the sum of hydrophobicities at positions 41 and 42. B, correlation with the sum of -sheet propensities at positions 41 and 42. C, correlation with the aggregation propensity predicted by the equation of Chiti et al. (26).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Role of Hydrophobicity—Fig. 1A shows that mutants with hydrophobic residues at both positions 41 and 42 have high propensities to aggregate. Some of these hydrophobic mutants, such as Val⁴¹-Ile⁴² and Leu⁴¹-Ile⁴², are even more prone to aggregate than the wild type (Ile⁴¹-Ala⁴²) sequence. The greater hydrophobicity of Ile relative to Ala at position 42 presumably accounts for this enhanced aggregation. Likewise, for the Val⁴¹-Ala⁴² mutant, the lower hydrophobicity of Val relative to Ile at position 41 may account for the slightly decreased aggregation of this mutant relative to wild type.

Mutants that have one hydrophilic and one hydrophobic residue at the C terminus (e.g. Lys⁴¹-Leu⁴², Gln⁴¹-Leu⁴², and Glu⁴¹-Leu⁴²) are slightly less prone to aggregate than the double hydrophobic sequences, whereas mutants that have two polar residues at the C terminus (e.g. Asp⁴¹-Glu⁴², Asp⁴¹-Gln⁴², and His⁴¹-Asn⁴²) are the least aggregating sequences in the library.

These results indicate that the hydrophobicity of residues at positions 41 and 42 is a major contributor to the enhanced amyloidogenicity of A42 relative to A40, and would be consistent with residues Ile⁴¹ and Ala⁴² occurring in the buried core of A fibrils.³

-Sheet Propensity—Although the hydrophobicity of residues at positions 41 and 42 clearly plays a dominant role in promoting aggregation, hydrophobicity alone does not explain the properties of all mutants in our collections. For example, four libraries of mutants were constructed in which only position 41 or 42 was randomized, whereas the other position was constrained to be either proline or glycine (TABLE TWO). For all four of these libraries, the vast majority (90%) of mutants yielded green fluorescent (i.e. less aggregating) GFP fusions (TABLE TWO), and the few white colonies that were observed did not express the fusions protein (presumably because of the presence of stop codons). These results suggest that all (or nearly all) sequences with proline or glycine at positions 41 or 42 have significantly reduced propensities to aggregate. This behavior was observed despite the fact that proline is a hydrophobic residue, and glycine is neither polar nor non-polar. Proline and glycine are known to be -sheet breakers (39), and their occurrence at positions 41 or 42 presumably interferes with the formation of cross- amyloid structure.

The wild type residues, Ile⁴¹ and Ala⁴², are both hydrophobic and good -sheet formers. In A42, these residues, together with residues Val³⁹ and Val⁴⁰ (which are also hydrophobic -sheet formers), may form a -strand at an early stage of aggregation, and thereby lower the kinetic barrier for the aggregation of A42 relative to A40.

Tertiary Interactions—Although hydrophobicity and -sheet propensity account for most of the observations reported here, two of the mutants shown in Fig. 1, Ile⁴¹-Arg⁴² and Arg⁴¹-Arg⁴², yield less fluorescent (i.e. more aggregating) GFP fusions than would be expected for sequences containing arginine, which is an extremely polar residue (24). The behavior of these mutants might be explained by the structure of A fibrils modeled by Tycko and co-workers (45) from solid-state NMR experiments. In that structure, A forms a hairpin, which would place the C-terminal amino acids in close proximity to glutamic acid at position 11. Interaction between the positively charged arginine and the negatively charged glutamic acid might compensate for burying the charged arginine side chain. This interpretation would suggest that a sequence with lysines at positions 41 and 42 might also yield less fluorescent (more aggregating) GFP fusions than expected for sequences containing charged residues at both positions. To test this expectation, we used site-directed mutagenesis to construct the Lys⁴¹-Lys⁴² mutant. Fluorescence of the GFP fusion of this mutant, although not as diminished as the Arg⁴¹-Arg⁴² mutant, was indeed lower than expected for a doubly charged dipeptide. (Its fluorescence was approximately midway between those of Asp⁴¹-Glu⁴² and Arg⁴¹-Arg⁴²; data not shown).

Correlation of Aggregation with Hydrophobicity, -Sheet Propensity, and Previous Models—To verify the importance of hydrophobicity and -sheet propensity at positions 41 and 42, we plotted the fluorescence of A42-GFP fusions against each of these parameters. As shown in Fig. 5A, the fluorescence of our mutants and the sum of hydrophobicities at positions 41 and 42 exhibit a strong negative correlation (R = 0.83). Thus, sequences having hydrophobic residues at these positions have higher propensities to aggregate.

Fluorescence of the A42-GFP fusions was also plotted against the sum of the -sheet propensities (25) at positions 41 and 42. As shown in Fig. 5B, this plot also showed a strong negative correlation (R = 0.85), thereby supporting the hypothesis that -sheet propensity at these positions favors aggregation.

Chiti et al. (26) showed that rates of aggregation could be predicted from intrinsic factors including hydrophobicity, -sheet propensity, -helical propensity, and charge. They derived an equation to quantify this prediction (see Equation 1 under "Materials and Methods").

For our collections of mutants in residues 41 and 42, we plotted the observed fluorescence of the A42-GFP fusions against the aggregation rate predicted by Equation 1 (Fig. 5C). The observed fluorescence and the aggregation predicted by this equation, which includes both hydrophobicity and -sheet propensity, showed a better correlation (R = 0.911) than either hydrophobicity alone (Fig. 5A) or -sheet propensity alone (Fig. 5B).

The Arg⁴¹-Arg⁴² mutant deviated significantly from the trend (Fig. 5C). As described above, the behavior of this sequence may be explained by attractive electrostatic interactions between the arginine side chain and glutamic acid at position 11.

Sequence Determinants of the Enhanced Amyloidogenicity of A42 Relative to A40—Although both A40 and A42 assemble into amyloid fibrils, the longer peptide aggregates more readily both in vitro and in vivo (17, 29, 44). Although it might have been hypothesized that length per se is sufficient to increase the amyloidogenicity of A, this hypothesis is disproved by our results. In particular, fluorescence studies of A42-GFP fusions containing polar amino acids at positions 41 and 42 demonstrate that these sequences are in fact less prone to aggregate than the null mutant, A40, which has both residues deleted entirely (Fig. 1).

Our experiments using several libraries of A42-GFP fusions as well as synthetic 42 residue peptides demonstrate that rather than length, it is the hydrophobicity and -sheet propensity of residues 41 and 42 that cause the enhanced amyloidogenicity of A42 relative to A40.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 609-258-2901; Fax: 609-258-6746; E-mail: hecht{at}princeton.edu.

² The abbreviations used are: A, amyloid ; GFP, green fluorescent protein; HFIP, hexafluoroisopropanol; HPLC, high pressure liquid chromatography; ThT, thioflavin T.

³ R. Tykco, personal communication.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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