Clustering and Spatial Arrangement of \xce\xb2-Sheet Sequence, but not Order, Govern \xce\xb1-Synuclein Fibrillogenesis\xe2\x80\xa0
The intrinsically unstructured protein \xce\xb1-synuclein (aS) is prone to misfold into cytotoxic \xce\xb2-sheet-rich oligomers and amyloid fibrils that underlie the pathogenesis of Lewy body diseases such as Parkinson\'s disease. An important, recognized fibrillogenesis parameter is amino acid content, whereas the influence of amino acid sequence distribution is not as well understood. The fibril core of aS encompasses five regions of high \xce\xb2-sheet propensity, termed \xce\xb21-\xce\xb25. Using four aS variants with identical amino acid composition but rearranged pseudorepeat motifs, we show that \xce\xb22-\xce\xb25 sequence clustering, but not order, is important for efficient fibrillogenesis. For molecular species progressing towards the fibrillar state, order invariably increases; i.e., the spatial arrangement of sequence elements becomes restricted. By introducing disulfide bonds in a fibril structure-based manner, it is demonstrated that a successful protofibril-to-fibril conversion is dependent upon the spatial arrangement of sequence elements of high \xce\xb2-sheet propensity. Moreover, a disulfide-linked aS dimer is shown to fibrillize rapidly. We propose that a conformational search underlies the emergence of a fibrillar aS nucleus that is directed by gaps in sequence between \xce\xb2-sheet regions and the accessible range of spatial \xce\xb2-sheet arrangements in soluble, prefibrillar oligomers. Based on the universal cross-\xce\xb2 sheet structure of amyloid fibrils, these principles are expected to apply to a wide range of amyloidogenic proteins.
<!>Molecular biology and protein production<!>Fibrillization kinetics, electron microscopy and CD spectroscopy<!>Role of \xce\xb2-sheet sequence clustering and order in aS fibrillogenesis<!>Role of \xce\xb2-sheet spatial arrangement in aS fibrillogenesis<!>Discussion
<p>In the course of the last century, there has been a significant increase in the recognition and prevalence of pathological conditions associated with improper protein deposition in humans (1, 2), perhaps in correlation to increased life expectancies. Pathological proteinaceous deposits have been associated with a number of diseases, most notably the prevalent neurodegenerative disorders (3-5). Protein deposits implicated in dementia are rich in predominantly one of three protein – α-synuclein, β-amyloid and tau – all of which are intrinsically unstructured, but nevertheless deposited as ordered fibrils (3-7). Consequently, an undesired folding pathway can be populated by these proteins, which leads to conversion of soluble monomeric proteins into insoluble amyloid fibrils. This pathway involves the formation of small oligomers (dimers, trimers, etc.) that assemble into soluble protofibrils, which eventually transition into insoluble fibrils (4-7). The structures of all examined amyloid fibrils contain a core region of cross-β-sheet structure, and soluble oligomers generally exhibit a significant content of β-sheet structure (4, 7, 8). This suggests that interactions between β-sheet-rich protein conformations give rise to the initiation, maturation and propagation of fibrillar misfolding in a rather universal manner. It has even been proposed that such self-propagating proteins, capable of storing information in a particular β-sheet arrangement, represented the first functioning biomolecules (9).</p><p>The misfolding of α-synuclein (aS)1 has been implicated in the demise of dopamineric neurons of the substantia nigra, which is associated with the phenotype of Parkinson's disease (5, 10). The sequence of the 140-residue protein aS comprises seven 11-residue degenerate repeats succeeded by a tail of low hydrophobicity and high net charge (Figure 1A and Figure S5 of the Supporting Information). The pseudorepeat sequence has amphiphilic character (Figure 1A-B), reminiscent of apolipoproteins (11), and allows aS to reversibly bind synaptic vesicles (11, 12), which, in turn, may inhibit neurotransmission (13). Within small variations between aS fibril preparations, deposited aS fibrils exhibit a rigid core encompassing residues 37-95 (14-17), which implicates the amphiphilic pseudorepeat sequence in misfolding. Within this sequence, which places aS outside of the typical charge-hydrophobicity phase space of intrinsically unfolded proteins (18), a 35-residue segment (residues 61-95), termed the NAC (non Aβ-amyloid component) region, had early on been implicated in neurodegeneration (19). An isolated peptide corresponding to the NAC region is capable of fibrillizing (20). However, aS fibrillogenesis is modulated by interactions that virtually involve its entire sequence and can even be abrogated despite the presence of the NAC sequence (21-25). Thus, while distinct sequence motifs are important for fibrillogenesis, they can be context dependent. It was further shown that protein mean charge, mean hydrophobicity, and mean β-sheet propensity allow fibrillization predictions of considerable accuracy for many proteins, including wild-type aS and mutant forms incorporating amino acid changes (22, 26). Combining the evaluation of such features in computational approaches that often consider β-sheet pairing possibilities and/or sequence motifs also led to useful predictions of the fibrillization propensity of amino acid sequences (27-31). In contrast to amino acid content and certain sequence motifs, the influence of amino acid sequence distribution and the role of the spatial arrangement of sequence elements on fibrillogenesis are not as well understood and are the focus of the current study.</p><p>The influence of amino acid sequence distribution can be examined by studying sequence-rearranged aS variants. An additional advantage to studying such variants is that a constant amino acid content is maintained; i.e., mean physicochemical properties are kept identical to the wild type. A recent study of this nature revealed that an aS sequence rearrangement based on interactions with several aggregation-inhibiting small molecules can abolish fibrillogenesis in apparent correlation to the fragmentation of β-sheet sequence propensities (32). The general significance of this observation is examined here in more detail by studying four specific rearrangements of aS pseudorepeats to evaluate the effects of β-sheet sequence clustering and order on fibrillogenesis. The progression of a molecular species along the fibrillization pathway invariably leads to an increase in its structural definition; an aS molecule transitions from a monomeric, unfolded state to the fibrillar, folded state, where it contributes to nucleating a fibril core, or joins an existing cross-β sheet arrangement. The structural determinants that govern the transitions between the defined higher order aggregates (e.g., the protofibril-to-fibril transitions) are unknown. To provide further insight into the mechanism of pathological aS misfolding, we examined six engineered intra- or intermolecular disulfide-mediated sequence restrictions that were based on the aS fibril structure (15), thereby enabling us to study the influence of β-sheet spatial arrangement on fibrillogenesis.</p><!><p>The genes of four aS variants with shuffled pseudorepeats, termed SaS-B to -E (Figure 1C and Figure S1 of the Supporting Information), were assembled from overlapping synthetic oligonucleotides by PCR (33), and were subcloned into the pET44 expression vector (Novagen, Inc.). Six disulfide-restricted variants were generated by altering selected codons in the wild-type aS expressing pRK172 vector to code for cysteine, using QuikChange mutagenesis (Stratagene, Inc.). One variant incorporated an intermolecular disulfide bond at residue A11, termed aS(11)-aS(11), and one variant incorporated an intramolecular disulfide bond at A11C-V26C, termed aS(11-26). Four variants incorporated an intramolecular disulfide bond that pairs T81C with V63C, T64C, N65C or V66C, termed aS(63-81), aS(64-81), aS(65-81) and aS(66-81).</p><p>For all variants as well as wild-type aS and β-synuclein (bS) overexpression in E. coli and protein purification were performed as described previously for the wild type (32, 34). However, variants containing cysteines were lysed by heat shock under reducing conditions (10 mM β-mercaptoethanol, 1 mM EDTA and N2 atmosphere). Prior to protein purification, disulfide bond formation was allowed to take place by dialyzing out the reducing agent overnight at 4 °C. This resulted in a range of disulfide-linked aS oligomers. To obtain the desired disulfide-linked monomeric or dimeric protein species (intraor intermolecular linkage), the covalently linked oligomers were separated by gel filtration (Figure S2 of the Supporting Information), employing a Sephacryl S100HR 26/60 column (GE Healthcare, Inc). Electrospray mass spectrometry confirmed the expected mass of the disulfide-linked variants (Table 1) and sample purity is illustrated by SDS-PAGE (Figure S2 of the Supporting Information).</p><!><p>Thioflavin T fluorescence was monitored to measure the emergence of amyloid fibrils as a function of time (35, 36). To remove previously formed aggregates, protein solutions were passed through a 100 kDa cut-off filter (Sartorius Stedim Biotech GmbH) immediately prior to use. This resulted in the absence of protofibrils and fibrils as evidenced by electron microscopy (Figure S3 of the Supporting Information). In the absence of fibrils that are capable of directly seeding new fibrils (37), thioflavin T fluorescence monitors the emergence of amyloid fibrils (35, 36) from the monomeric 14-kDa proteins and any potential residual amount of small oligomers. However, when using aS protein preparations of different ages, little differences in fibrillization kinetics were found (Figure S4 of the Supporting Information), suggesting that the amount of residual small oligomers was constant and/or small.</p><p>A thioflavin T (Calbiochem, Inc.) stock solution was prepared at 5 mM in MilliQ-H2O and filtered through a 0.2 μm cellulose acetate filter. Protein fibrillogenesis was evaluated at a protein concentration of 100 μM in 25 mM NaH2PO4/Na2HPO4, pH 7.4, 0.02% NaN3 solution. Samples were incubated at 37 °C in upright 1.5 ml Eppendorf tubes under circular agitation (3 mm stroke, 1,200 rpm), using an Eppendorf Thermomixer. Thioflavin T fluorescence was measured at selected time points (Figures 3 and 5B) by removing a constant volume from the incubated sample, diluting it with 50 mM Glycine·NaOH, pH 8.5 solution, and adding thioflavin T to yield final concentrations of 5 μM protein and of 25 μM thioflavin T, respectively. Fluorescence measurements were immediately carried out on a Varian Cary Eclipse fluorimeter by exciting at 446 nm (5 nm slit width) and scanning the emission wavelength from 450 to 550 nm (20 nm slit width). Fluorescence intensities at 482 nm were plotted as a function of time after subtracting the buffer signal and fitted to a sigmoidal function.</p><p>For electron microscopy, 10 μl of the incubated sample was taken after 168 hr, placed on carbon-coated copper grids (Electron Microscopy Sciences, Inc.) for 10 min, and stained with 2% uranyl acetate for 1 min. Images were taken on a Jeol JEM 1400 electron microscope operated at 100 kV. CD measurements were carried out at 25 °C on a JASCO J-810 spectropolarimeter using 20 μM of 100 kDa cut-off filtered protein in 10 mM KH2PO4/K2HPO4, pH 7.4, solution. For each sample, eight scans were accumulated, averaged and corrected for the buffer signal.</p><!><p>Based on backbone amide proton H/D exchange and secondary 13Cα/13Cβ chemical shifts, five β-strands, β1-β5, encompassing residues 37-43, 52-59, 62-66, 68-77 and 90-95, respectively (Figure 1A), were recently assigned to the aS fibril core (15). Overall, these definitions are in good agreement with the β-sheet propensities of the aS sequence (Figure 2A), and we use β1-β5 to denote both β-sheet propensities and actual β-sheet structure. Furthermore, intramolecular β1-β2, β2-β3, β3-β4 and β4-β5 interactions were postulated to contribute to the formation of five layers of parallel, in-register β1-β′-β″, β2-β2′-β2″, etc. sheets between aS, aS′ and aS″ molecules to form cross-β structure (15). To assess the importance of the clustering and the order of β1-β5 strands in the context of an unaltered aS amino acid composition (mean physicochemical properties), four sequence-rearranged aS variants, termed SaS-B, -C, -D and -E, were prepared (Figure 1C). Although a number of rearrangement schemes may have been envisioned, we restricted ourselves to the swap of the 11-residue aS pseudorepeats (Figure 1B). In addition to producing changes in β-sheet propensity distributions (Figure 2), this concomitantly altered the distribution of charges and hydrophobicities (Figure 1B-C and Figure S5 of the Supporting Information).</p><p>Variant SaS-B exchanged repeat III at the beginning of the fibril core with repeat I outside of the core (Figure 1C) to reduce β-sheet clustering (Figure 2A,B). Nevertheless, fibrillization was similar to wild-type aS with a somewhat longer lag time (Figures 3 and 4A-B), indicating that fibrillogenesis proceeded without β1-β2 sequence proximity and perhaps also without β1-β′ cross-β interactions. Variant SaS-C, which has swapped repeats VI and IV (Figure 1A,C), failed to fibrillize under the same conditions (Figure 3). It resulted in the deposition of amorphous aggregates, as evidenced by a comparison to β-synuclein (bS) aggregates (Figure 4D-E). bS is non-amyloidogenic and exclusively forms amorphous aggregates (32, 38, 39). This indicated that the canonical aS fibrillization pathway had been disturbed. Despite having incurred only one repeat swap, β-sheet pairing possibilities, as well as hydrophobic and electrostatic sequence patterns, were significantly altered throughout β1-β4 (Figure 2C and Figure S5 of the Supporting Information). As a result, a new β-sheet and hydrophobic cluster comprised of β1/β4 was created at the expense of mainly β3. The decrease of the β3-sheet score in the context of the new sequence environment effectively increased the gaps between the β1/β4 and β2 regions compared to the wild type, resulting in the fragmentation of regions of high β-sheet propensity. Interestingly, the new β/β4 cluster, which exhibits a comparable β-sheet propensity and increased hydrophobicity compared to β3-β4 of wild-type aS, was unable to trigger SaS-C fibrillization. In SaS-D, the swap of repeats VI and I transferred β4 to the N-terminus, which also increased the fragmentation of β-sheet propensities (Figures 1C and 2C). Consequently, the absence of fibrillization and deposition of SaS-D as amorphous aggregates (Figure 3 and Figure 4F) was not surprising. An extensive fragmentation of β-sheet propensities compared to the wild type had also been noted for the non-fibrillizing variant SaS-A that had been studied earlier (Figure 1B-C and Figure S6 of the Supporting Information) (32, 34). Finally, the variant SaS-E implemented three repeat swaps (Figure 1C) that were devised to rearrange β-sheet propensities without introducing new gaps, except for β1 (Figure 2B). Based on the fibrillizing nature of SaS-B, β1-sheet propensities were allowed to be dispersed. The SaS-E sequence configuration, which preserved the distribution of hydrophobicity and charge well relative to wild-type aS (Figure S5 of the Supporting Information), readily fibrillized with, again, a somewhat longer lag time than the wild type (Figures 3 and 4C). In conclusion, within the context of unchanged mean physicochemical molecular properties, fibrillizing SaS-B and SaS-E show promiscuity and redundancy in wild-type aS β-sheet sequence order and pairings, whereas SaS-C, SaS-D and SaS-A signify a limited tolerance for gaps in sequence between regions of high β-sheet propensity and perhaps hydrophobicity as well.</p><!><p>In the course of aS misfolding, a dimeric species is detectable (32, 40), suggesting that certain interactions between members of the dynamically unfolded aS structural ensemble (41, 42) can give rise to such a misfolded species, which may be viewed as the first step of the fibrillization pathway. Similarly, the transition to higher-order structures (e.g., the protofibril-to-fibril transition) may depend on the accessible structures of constituent molecules, most notably β-sheet arrangements, since aS amyloid fibril formation results in defined cross-β sheet structure (14, 15, 17). Here, we investigated whether transitions to higher-order oligomers can be favored, or disfavored, upon restricting β-sheet spatial arrangements in a fibril structure-based manner. To bias the structural ensemble of aS, disulfide bonds can be introduced (43). This approach was employed to restrict the aS structural ensemble around the region of highest predicted β-sheet propensity, encompassing strands β3-β4 (Figure 2A). Four disulfide-restricted variants were prepared by pairing T81C, located in the β4-β5 connector, with V63C, T64C, N65C and V66C, respectively, of strand β3 (Figure 5A), termed aS(63-81), aS(64-81), aS(65-81) and aS(66-81), respectively. Since the side chains of two consecutive residues of a β-sheet point in an opposite direction, at most two out of the four disulfide variants were expected to efficiently fibrillize into the canonical cross-β sheet arrangement of wild-type aS. Moreover, the closer the disulfide pairings become in sequence, the more disturbances in intramolecular β3-β4 stacking would ensue (Figure 5A). This would lead to a less compact fibril core, unless compensated by additional rearrangements involving β1, β2 and β5. For comparison, the A11C-V26C pairing, termed aS(11-26), outside of the fibril core was studied (Figure 2A). Moreover, A11C was used to generate a disulfide-linked dimer, termed aS(11)-aS(11), which may accelerate the sampling of intermolecular interactions leading to aS misfolding. It is noted that all amino acid substitutions did not change the aS charge distributions, and altered amino acid hydrophobicities and β-sheet propensities in a conservative manner.</p><p>As expected, not all T81-paired variants fibrillized (Figure 5B); only aS(63-81) produced fibrils (Figures 5B and 6A). For aS(64-81), aS(65-81) and aS(66-81), the predominantly observed species were protofibrils (Figure 6B-D), showing that the protofibril-to-fibril transition was blocked, whereas initial β-sheet assembly was efficient enough to largely avoid unspecific, amorphous aggregation (Figure 7). It is also noted that aS(63-81) and aS(66-81) have substituted the same residue types (valine and threonine), thus excluding any difference in misfolding behavior due to a change in mean protein physicochemical properties. The permissive range of β-sheet spatial arrangements in protofibrils therefore appears larger than in fibrils. The aS(11-26) variant unexpectedly produced fibrils very slowly despite its cysteine pairing outside of the fibril core (Figures 2A, 5B and 6E), allowing a dominant amount of amorphous aggregation to occur (Figures 5B and 6E). Apparently its initial oligomerization was relatively inefficient (Figure 7). As expected, the aS(11)-aS(11) dimer fibrillized rapidly. In addition to more "conventional" elongated fibrils, it also produced small linear and dense spherical particles (Figure 6F). The lack of any significant fibrillization lag time (Figure 5B) suggested that aS(11)-aS(11) was able to rapidly initiate the formation of molecular species along the fibrillization pathway. Intermolecular disulfide-linked aS avoids the entropic penalty of dimer formation (from monomeric aS) and increases the local aS concentration, accelerating the rate of aS-aS encounters. In conclusion, the ability of aS(64-81), aS(65-81) and aS(66-81) to form protofibrils, but, unlike aS(63-81), not fibrils, demonstrates that a successful protofibril-to-fibril conversion is dependent upon the accessible spatial arrangement of sequence elements of high β-sheet propensity.</p><p>Finally, it may be expected that the introduced spatial restrictions affected the ensemble of dynamically unstructured protein conformations. The far-UV CD spectra of aS(64-81), aS(65-81) and aS(66-81) were similar to wild-type aS, but aS(11-26), aS(63-81) and aS(11)-aS(11) were shifted towards more positive mean ellipticities (Figure 5C). Although all CD spectra were characteristic of proteins exhibiting dynamically unstructured ensembles (44), this difference, which is present at all wavelengths, indicated a shift in underlying ensemble conformer distributions relative to the wild type. It would be compatible with an increase in "extended" protein backbone conformations (ellipticity increase at 198 nm; Figure 5C). Albeit interesting to note, these observations are nevertheless difficult to interpret in terms of fibrillization propensity because only aS(63-81) and aS(11)-aS(11), but not aS(11-26), fibrillized efficiently.</p><!><p>Central to developing therapeutic approaches aimed at neutralizing the pathological species formed along the fibrillar misfolding pathway (3, 4, 6, 7) is a detailed understanding of the fibrillar aggregation mechanism. By analyzing the fibrillization kinetics and morphologies of four sequence-rearranged aS variants with identical mean physicochemical properties, we have shown that fibrillogenesis is tolerant to the order of sequence elements of high β-sheet propensity, but not to their fragmentation. In other words, cross-β sheet pairings and intramolecular β-sheet stacking are promiscuous in aS fibrils. Based on the observation of different filament morphologies between aS mutants of differing mean physicochemical properties, it had been noted that multiple molecular interactions can underlie the fibril assembly of aS (22). In addition, different fibrillar β-sheet arrangements were observed in wild-type aS and its aS(A53T) variant (45). Nevertheless, the extent of the sequence promiscuity revealed in the present study is perhaps surprising. In variant SaS-E, 24 out of 59 non-equivalent fibril core residue positions were exchanged (Figure S1 of the Supporting Information), but fibrillization was nonetheless highly efficient. In marked contrast, fibrillization kinetics were sensitive to an increase in gaps in sequence between regions of high β-sheet propensity. Considering the elevated β-sheet content of early aS oligomers (46, 47), this suggests that gaps constitute a kinetic barrier to "finding" a fibril-nucleating species before aggregation proceeds in an unspecific, amorphous manner (Figure 7). This is reminiscent of the concentration dependence of forming either fibrillar or amorphous aggregates in the amyloid-β peptide Aβ40 (48). Thus, although the fibrillar state of the rearranged aS variants may still be lower in free energy than the monomeric or unspecifically aggregated states, finding an avenue to the fibrillar state appears to be inefficient. This is analogous to the fact that even non-amyloidogenic β-synuclein can be forced to fibrillize under persuasive enough conditions (49), and perhaps this applies to most proteins.</p><p>Molecular dynamics simulations suggested that hydrophobic clustering precedes β-sheet formation in the fibrillar misfolding pathway (50). Regions of high, continuous hydrophobicity may therefore favor fibrillization and their fragmentation may contribute to the absence thereof. Indeed, aS sequence regions of elevated hydrophobicity generally correlate well with regions of high β-sheet propensity (Figure 2A and Figure S5 of the Supporting Information). However, fibrillization can also be suppressed despite the presence of a large hydrophobic cluster, and fibrillization can be maintained despite the elimination of such a cluster. The SaS-C variant created the highest consecutive hydrophobic sequence among all studied variants, including wild-type aS (Figure S5 of the Supporting Information), but nevertheless failed to fibrillize. The deletion mutant aS(Δ73–83), which does not introduce new gaps between β-sheets, eliminates an extended hydrophobic region in the NAC region (Figure S6C of the Supporting Information), but still fibrillizes, albeit not as well as wild-type aS (22). In other words, aS(Δ73–83) still allows successful β-sheet pairings, further illustrating redundancy and promiscuity in such pairings. Thus, hydrophobicity plays an important role in fibrillogenesis, but the distribution of β-sheet sequence appears decisive in the case of aS. Similarly, the distribution of charged residues (Figures S5-S6 of the Supporting Information) will be able to modulate fibrillization kinetics.</p><p>By analyzing the fibrillization kinetics and morphologies of four disulfide-restricted aS variants, enclosing β-sheet propensities β3-β4 (Figures 1A and 2A), we further demonstrate that a successful protofibril-to-fibril transition is dependent on the accessible range of spatial β-sheet arrangements (Figure 7). It is noteworthy that the aS fibrillization pathway can be dissected in a manner that preserves the efficient formation of protofibrils, but selectively prevents fibril formation. Further examples of such behavior are the deletion mutants aS(Δ71–82), aS(Δ74-79) and aS(Δ76-77) (51, 52), which involve residues of sheet β4 (Figure 1A) and therefore cannot fibrillize in the canonical cross-β sheet arrangement of the wild type (Figure 5A). These sequence deletions are in close proximities to the disulfide pairings between sheet β3 and the β4-β5 connector made in the present study (Figures 2A and 5A), attesting to the importance of the spatial β3-β4 arrangement in fibril assembly. In this context, we also note the ability of a 9-42 and 69-89 double disulfide-restricted aS variant to fibrillize (43). These residue pairings contain different and more intervening residues than were enclosed by the restrictions made here, which, in turn, may permit larger deviations from the canonical fibril structure of the wild type.</p><p>Two additional disulfide-restricted aS variants were studied. The aS(11-26) variant fibrillized, but did so slowly. This is yet another example of a sequence alteration outside of the fibril core that can disturb fibrillization (21, 25). Since aS(11-26) accumulates mainly as amorphous aggregates (Figure 5B), the disulfide restriction appeared capable of biasing the structural ensemble in a manner that complicated early β-sheet interactions (Figure 7). The high preference of several aggregation-inhibiting small molecules for interacting with the region encompassing residues 3-18 and 38-51 is noted in this context (34, 53). Beyond the amphiphilic nature of many aggregation-inhibiting molecules (34), the often multivalent character of these molecules, which are also capable of forming micellar aggregates that bias aS conformers but do not induce folding (34, 54), may aid in hindering fibril assembly by restricting the range of accessible β-sheet arrangements. Such a mechanism would be supported by their ability to largely inhibit fibrillization, but not oligomerization (40). Finally, the rapid fibrillization kinetics of the intermolecular aS(11)-aS(11) dimer provided further strong evidence that access to the fibrillization pathway is kinetically controlled. In other words, a conformational search underlies aS amyloid misfolding, and the aS(11)-aS(11) disulfide linkage accelerates the sampling of intermolecular interactions.</p><p>In summary, the fibrillization kinetics of the ten aS variants examined in the present study indicate that a conformational search underlies the emergence of a first fibrillar aS species. The search efficiency for such a fibril nucleus is sensitive to gaps in sequence between β-sheet regions and the accessible range of spatial β-sheet arrangements at the protofibril stage. Based on the universal cross-β-sheet structure of amyloid fibrils (4, 7, 8), these principles are also expected to apply to the fibrillogenesis of other amyloidogenic proteins. However, highly Asn/Gln-enriched proteins, whose fibrillization strongly depends on the content of these amino acids (55), may represent a notable exception to this statement. A relatively inefficient, but sequence tolerant search-driven folding pathway may have contributed to the formation of early proteinaceous biomolecules such as amyloids (9).</p>