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Complex System Assembly Underlies a TwoTiered Model of Highly Delocalized Electrons Miguel Mompeán, Aurora Nogales, Tiberio A Ezquerra, and Douglas V. Laurents J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b00699 • Publication Date (Web): 03 May 2016 Downloaded from http://pubs.acs.org on May 4, 2016

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The Journal of Physical Chemistry Letters

Complex System Assembly Underlies a Two-Tiered Model of Highly Delocalized Electrons Miguel Mompeán1,*, Aurora Nogales2, Tiberio A. Ezquerra2 and Douglas V. Laurents1,* 1 2

Instituto de Química Física “Rocasolano” (IQFR-CSIC), Serrano 119, Madrid E-28006, Spain. Instituto de Estructura de la Materia (IEM-CSIC), Serrano 121, Madrid E-28006, Spain. Correspondence: [email protected], [email protected]

KEYWORDS: Amyloid; Electrical conductivity; Density Functional Theory; Cooperative Hydrogen Bonds; Electron delocalization

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ABSTRACT Amyloid fibrils are exceptionally stable oligomeric structures with extensive, highly cooperative, H-bonding networks whose physical origin remains elusive. While nonpolar systems benefit from both H-bonds and hydrophobic interactions, we found that highly polar sequences containing glutamine and asparagine amino acid residues form hyperpolarized H-bonds. This feature, observed by DFT calculations, encodes the origin of these polar oligomers’ high stability. These results are explained in a theoretical model for complex amyloid assembly based on two different types of cooperative effects resulting from highly delocalized electrons, one of which is always present in both polar and hydrophobic systems. Experimental electric conductivity measurements, ThT fluorescence enhancement and NMR spectroscopy support this proposal and uncover the conditions for disassembly.

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Amyloids are protein oligomers stabilized by weak, non-covalent interactions such as H-bonds, van der Waals, and the hydrophobic effect. It is generally believed that the latter make the chief contribution to amyloid and protein stability since protein/protein and protein/solvent H-bonds are thought to be energetically similar in stability,1 and other types of interactions are less abundant. Nevertheless, the study of proteins carrying mutations designed to disrupt native H-bonds supports that they substantially stabilize globular protein and amyloid structures.2,3 Further evidence is the discovery of highly polar amyloids with numerous H-bonds and very few hydrophobic components,4 yet the basis of their stability is still unclear. A very interesting property from the physical point of view is the mutual strengthening experienced by H-bonds in H-bonding networks. This so-called hydrogen bond cooperativity (HBC)5–12 might be responsible for providing amyloids with unexpected features of electron-delocalized systems, such as intrinsic fluorescence and electric conductivity.13,14

Here, we have analyzed the source of cooperative H-bond effects to understand amyloids’ remarkable stability and elucidate the origin of their unexpected charge-transfer properties. Both polar and hydrophobic fibrils share a common architecture in which the monomers adopt extended conformations (β-strands) and are stabilized by a large set of inter-monomer main chain–main chain Hbonds (Figure 1A), which we call backbone–backbone (bb–bb) H-bonds (Figure 1B). This arrangement of sequentially stacked β-strands is called a β-sheet. Interestingly, in glutamine- and asparagine-rich (Gln/Asn-rich) sequences which lack strong hydrophobic contributions there are additional H-bonding networks formed by the stacking of these solvent-exposed residues’ side chains into ladders extending out from the fibril axis (side chain–side chain or sc–sc H-bonds, Figure 1B).4,15 We first searched for differences in bb–bb vs. sc–sc H-bonds by performing the set of Density Functional Theory (DFT) calculations depicted in Figure 2A on four different systems; namely, (i) the seven-residue GNNQQNY segment from the yeast prion Sup354, (ii) a mutated version (termed polyGly) in which all sc–sc contacts are removed, while keeping bb–bb interactions, (iii) a highly

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hydrophobic segment corresponding to residues 17–42 of the Aβ peptide involved in Alzheimer’s disease (AD)16, and (iv) a shorter six residue segment: MVVGGVV, corresponding to Aβ35-4017 due to the prohibitively large size of Aβ17–42 to perform the full set of calculations.

We calculated the interaction energies between selected pair of monomers (β-strands), which we called Hydrogen Bonding Strength (HBS) throughout this work, as they provide a direct quantification of the H-bonding10 (See Computational Methods). The HBS values were evaluated in three sets differing in the exterior/interior nature of their β-strands: whereas set 1 are edge β-strands, sets 2 and 3 have β-strands on at least one side, respectively (Figure 2A). Compared to previous works,8–10 which limited their analysis to edge β-strands, our study also probes how the HBS of internal β-strands varies as the oligomer grows. As described below, this analysis reveals an important difference in HBC between Q/N-rich and hydrophobic amyloids. An incoming monomer docking the preformed β-sheet always interacts with higher HBS values, as expected in cooperative H-bonds.5–12 In contrast, when this magnitude is computed for any given pair of β-strands (black, red or green), two different results are obtained for polyGly and the Aβ segments with respect to the Q/N-rich oligomer. In hydrophobic fibrils (Aβ35-40, Aβ17-42) and in polyGly, no significant changes are observed within each set of values despite the length of the β-sheets. However, these interactions become stronger and stronger in GNNQQNY, as indicated by the decreasing black, red and green curves (Figure 2B,C). Based on these observations, we propose that the overall cooperativity (HBC) arises from two different contributions (Eq. 1):

HBC = bas-HBC + hyper-HBC

(1)

There is a ubiquitous term, which we call basal HBC (bas-HBC) that is common to all amyloids regardless of their polar or hydrophobic nature. It arises from bb–bb H-bonds, which are always present 4


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(Figure 1A,B). We propose that Gln/Asn-rich sequences possess a second type of HBC called hyperpolar HBC (hyper-HBC). Since this contribution vanishes upon removal of the Gln/Asn side chains (Figure S1 in the SI), and is not present in the hydrophobic Aβ segments, the hyper-HBC might contribute to the self-assembly of Gln/Asn-rich polar segments containing few hydrophobic moieties. To ascertain this hypothesis, we considered two additional factors that might originate this effect; namely, the effective number of H-bonds, considered as the sum of sc–sc and bb–bb interactions, and the parallel or antiparallel monomer configuration. Whereas GNNQQNY and Aβ35–40 are composed by roughly the same number of residues (7 and 6, respectively), GNNQQNY is able to form five additional H-bonds due to the amide groups of its Asn and Gln side chains. Therefore, two monomers of GNNQQNY are mutually bound by 11 H-bonds, while only 5 are present in Aβ35–40. Moreover, they differ in their parallel (GNNQQNY) and antiparallel (Aβ35–40, MVGGVV) orientations. To test whether these differences contribute to the existence of hyper-HBC we considered the Aβ17–42 oligomer, where the monomers assemble in parallel like GNNQQNY, but form a larger number (22) of H-bonds (Figure S2 in the SI). The fact that only bas-HBC is observed in Aβ35–40, Aβ17–42 and polyGly, suggests that the second contribution to the HBC arises from Gln and Asn amino acid residues, and does not depend on monomer length, number of H-bonds, or orientations (Figures S1 & S2). The inclusion of vdW corrections results in more stabilizing energies and does not alter the two contributions to the overall HBC observed, which has already been suggested by Rossi et al.12 (Figure S3).

In order to obtain a molecular picture and correlate these observations with biophysical experiments, all the resulting Kohn-Sham DFT orbitals were transformed into more intuitive chemical representations using the Natural Bonding Orbital formalism.18,19 We observed that the cooperative effects in H-bonds arise from the N lone pair electron delocalizing onto an antibonding orbital with pure (~100%) p-character of its attached C=O, which results in an increased negative charge on the oxygen 5



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atom that will bind the incoming monomer more strongly (Figure 3A,B). This delocalization, common to both bb and sc CONH groups is the source of the ubiquitous bas-HBC (Figure 1). The amide groups’ ability to act as H-acceptors (A: C=O) and as H-donors (D: N-H) makes bb –CONH– groups able to behave as a conjugate –(···)–AD–Cα–AD–Cα(···)– chain mediated by the Cα linker. In this context, amide sc in Gln and Asn can be regarded as terminal groups, which can be visualized as -CONH–H, with the hydrogen atom in bold acting as a cap to block the conjugation that occurs in the main chain. In addition to the ubiquitous delocalization of the N lone pair to the C=O antibonding orbital (which is almost 100% p character), in amide sc the N lone pair also delocalizes onto a C=O antibonding orbital with mixed sp character (60% s, 40% p) –Table S1-. This feature is reminiscent of the non-bonding – antibonding interaction in proteins discovered few years ago using a similar DFT/NBO procedure.20 Here, it is interesting that these sp C=O antibonding orbitals do not become populated in bb CONH groups, neither in MVGGVV nor GNNQQNY, supporting the idea that Gln and Asn side chains play a special role. This particular delocalization and the capping H-atom in CONH2 that prevents electron delocalization out of the sc–sc H-bond, originate the hyper-HBC. This occurs because the delocalization of the N lone electron pair is concentrated on the C=O in -CONH–H, populating new orbitals, whereas in the case of backbone groups (-CONH–Cα–CONH–Cα–[···]) it is also spread over the chain onto antibonding orbitals such as those of N(i)-Cα(i) and N(i)-C’(i).

To validate these computational results, we measured the experimental conductivity of amyloid fibrils from GNNQQNY and Aβ17-42. We first prepared aged fibrils as described in Fig. S4 in the SI, and used broadband dielectric spectroscopy to obtain the dc component of the electric conductivity (σdc). We observed that σdc in H2O was two orders of magnitude higher in GNNQQNY than in Aβ17-42, which is in line with our computational results of an increased electron delocalization along sc–sc Hbonds with respect to bb–bb interactions. Accordingly, one promising strategy for fibril disassembly would be to disrupt the electron flow through the exposed side chains, by solvating them with a very 6


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strong H-bonding acceptor such as dimethyl sulfoxide (DMSO, Fig. S5). Our results show that in the presence of 100% DMSO the σdc values of GNNQQNY are lowered at values similar to those observed for Aβ17-42. Interestingly, these hydrophobic fibrils did not show notorious changes in the denaturizing solvent, as shown in Figure 4A. This is consistent with the burial of bb–bb H-bonds within the fibrillar core, which makes them more resistant to DMSO compared to the exposed sc–sc counterparts. To ascertain this assumption, Thioflavin–T (ThT) assays were performed on fibrils of both GNNQQNY and Aβ17-42 in DMSO. ThT is a conformational probe whose fluorescence is enhanced by binding to amyloid-like oligomers and very strongly enhanced by binding mature amyloid fibrils. 21 After one or seven days of incubation, the ability of the samples to enhance ThT fluorescence was measured and compared to that of parent samples in water. Whereas DMSO is an appropriate solvent to maintain Aβ monomeric,22 once the fibrils are well formed and have reached the maximum cooperative effects, they are more resistant to disruptive effect of 100% DMSO than the Gln/Asn-rich system, which readily dissociates after one day (Figure 4B). This is in line with previous observations on the hydrophobic α-synuclein amyloid fibrils,23 and supports the special role of Gln and Asn side chains in the highly efficient assembly of the polar oligomer. The lower σdc values of GNNQQNY in DMSO are compatible with dissociation resulting from solvent–side chain interactions disrupting native sc–sc Hbonds, as gauged from the ThT assays. For further corroboration, we performed solution NMR experiments, which provide atomic level information and do not require a reporter molecule such as ThT. Solution NMR readily detects soluble monomers, whereas larger oligomers and amyloid fibrils are invisible. In the case of Aβ17-42 fibrils incubated seven days in DMSO, only low-intensity signals were observed in the 1H spectrum, compared to samples of freshly dissolved monomer used as a control (Figure 4C). This is indicative of large oligomers or suspended fibrils (as detected by ThT). In contrast, in the GNNQQNY samples incubated one week in DMSO, monomers are definitely present, as revealed by 1D 1H spectra (data not shown) and a 2D 1H-1H TOCSY spectrum. These monomers seem to be in slow equilibrium with the amyloid7



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like species detected by ThT fluorescence since when the sample was transferred to D2O, its amide 1H’s quickly exchanged with solvent deuterons (Figure 4D).

In summary, we report two classes of H-bond cooperativity that stabilize the GNNQQNY fibrils, one of which arises from the singular NBO interactions of amide side chains that enables large electron delocalization and higher electric conductivity. The hydrophobic Aβ amyloid is also stabilized by the first class of H-bonding cooperativity, but not the second, suggesting how Gln and Asn-rich sequences compensate for the lack of hydrophobic interactions. The two-tiered model proposed seems to be independent of the number of H-bonds, and is supported by experimental results from dielectric and nuclear magnetic resonance spectroscopies and ThT recognition in water and DMSO. Future experiments will be necessary to test this model's generality to all amyloids. Nevertheless, it already offers an explanation for the surprising intrinsic fluorescence and charge-transport properties of these polymeric systems, as well as for the astonishing conformational stability of GNNQQNY that lacks strong hydrophobic contributions. Finally, these results open an avenue towards the design of inhibitors with potent H-bond accepting moieties that would act specifically on Q/N-rich amyloids, such as the polyglutamine expansions implicated in nine diseases, and the Q/N-rich stretch of TDP-43 tied to Amyotrophic Lateral Sclerosis.9

EXPERIMENTAL METHODS Fibril Formation. A peptide whose sequence, GNNQQNY, corresponds to Sup35(7-13) was purchased from Genescript (New Jersey, USA).

Its purity was over 95% as assessed by HPLC. NMR

spectroscopy and mass spectrometry were used to confirm the peptide’s composition. The peptide: LVFFAEDVGSNKGAIIGLMVGGVVIA, which corresponds to residues 17-42 of the Aβ peptide, was purchased from rPeptide. Fibrils were formed by dissolving each peptide to an initial concentration of 2 mM in mQ water followed by incubation at 25 ºC for more than three weeks. 8


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Broadband Dielectric Spectroscopy. Twenty mL of suspension containing GNNQQNY and Aβ17-42 fibrils in water or DMSO-d6 were used in each experiments. In the case of measurements in DMSO-d6, fibrils were lyophilized and dissolved in this solvent and they were directly measured without further incubation to minimize changes due to possible dissociation and subsequent contributions of monomeric species to the dielectric spectrum. Complex dielectric permittivity measurements (ε* = ε′ − iε″) were performed isothermally over a 10−1 < F/Hz < 106 frequency(F) range. The electrical conductivity, σ*, can be estimated as σ*=i2πFεoε* where εo is the vacuum dielectric permittivity.24 A Novocontrol system (Novocontrol Technologies GmbH & Co. KG, Germany) integrating an ALPHA dielectric interface was employed. The temperature was controlled by means of a stream of nitrogen gas (QUATRO from Novocontrol) with a temperature error of ±0.1º C during every single sweep in frequency. A dielectric liquid cell was used for the measurements. A complete depiction of the experimental procedure used to correlate experimental observations with our theoretical results is shown in Fig. S4 in the SI. ThT Assay. Aged fibrils of GNNQQNY and Aβ17-42 in water (vide supra) were lyophilized and dissolved in 600 mL DMSO-d6. After one or seven days of incubation in DMSO, centrifugation was used to settle fibrils and then aliquots were drawn from the supernatant. ThT was added to a final concentration of 50 µM to both parent samples and fibrils dissolved in DMSO, and the amyloid-induced enhancement of ThT fluorescence25 was measured on a Jobin-Yvon Fluoromax-4 instrument using 3 nm excitation and emission slit widths. The excitation wavelength was 440 nm and emission was recorded over 460-500 nm at a scan speed of 2 nm·s-1. A 1.0 mM stock solution of ThT (Sigma, St. Louis, USA) was prepared in 3mM KH2PO4/K2HPO4 buffer, pH 6.8. Nuclear Magnetic Resonance Spectroscopy. 1D 1H-NMR and 2D 1H-1H TOCSY (60 ms mixing time) NMR spectra of GNNQQNY and Aβ17-42 fibrils dissolved and incubated in DMSO-d6 for seven days were recorded at 30 ºC on a 600 MHz Bruker spectrometer equipped with a cryo-probe and z-gradients. 9



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To test for the presence of stable inter-peptide H-bonds, GNNQQNY fibrils in DMSO-d6 were lyophilized and diluted into D2O and an additional TOCSY spectrum was recorded. All the spectra were acquired and processed using the Bruker program Topspin version 2.1.

COMPUTATIONAL METHODS Quantum Mechanical Calculations. QM calculations were performed with the Gaussian09 software,26 The largest oligomers (pentamers, Supplementary Fig. S6) where optimized with Ca and non H-bonded side chains atoms fixed, while the amide moieties remained unrestrained to optimize the H-bonding interactions. The individual fragments for which interaction energies were computed (Fig. 2A) strictly have the same geometry as in the corresponding optimized pentamers to ensure that the relaxation effects of the structures are decoupled from the contributions to the overall cooperativity observed. This procedure prevents attractive strain8 and distortions with respect to the experimental structures determined at atomic-resolution used in this work, allowing a direct estimation of the contribution from the H-bonding interactions,10 so that theory and experiments can be compared. As for the level of theory, the M06-2X functional was chosen27 since it was derived for the computation of non-covalent interactions and has been successfully applied to biological systems.28,29 With respect to the basis set, the 6-31+G(d) was selected as a good balance between accuracy and computational cost to evaluate all the systems at the same level of theory and obtain reliable comparisons, considering the large number of atoms involved in these calculations. All the calculations were performed with a solvation continuum model (PCM), as implemented in the Gaussian09 package, to include the effect of solute polarization. The interaction energies were counterpoise (CP) corrected in gas-phase to account for basis set superposition error, assuming that the molecular orbitals do not change with the inclusion of PCM. This is a standard procedure, as CP cannot be used with implicit solvation. The D3 version of Grimme’s dispersion30 was applied to all the calculations, and the NBO analysis was performed with the NBO3.1 program,18 as implemented in Gaussian. The distinct stereoelectronic nature of the two electron lone 10


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pairs of the O atom was analyzed to ensure that the NBO transformation of the DFT Kohn-Sham orbitals is an appropriate descriptor of the H-bonded systems studied in the present work (Table S2).

ASSOCIATED CONTENT Supporting Information. Detailed experimental and computational procedures, two supplemental tables and six supplemental figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected]; phone: +34 91-745-9543 Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by Grants CTQ2010-21567-C02-02 (FPI Fellowship to MMG), SAF201349179-C2-2-R (DVL) and an EU JPND AC14/00037 (DVL), and Grants MAT2012-33517 and MAT2014-59187-R (TE and AN). We would also like to thank Dr. J. Cerezo from the ICCOM-CNR (Pisa, Italy) for his critical comments on this manuscript.

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21. Lindgren. M.; Sörgjerd, K.; Hammarström, P. Detection and Characterization of Aggregates, Prefibrillar Amyloidogenic Oligomers, and Protofibrils Unsing Fluorescence Spectroscopy. Biophys. J. 2005, 88, 4200–4212. 22. Laurents, D. V.; Pantoja-Uceda, D.; López, L. C.; Carrodeguas, J. A.; Mompeán, M.; Jiménez, M. Á.; Sancho, J. DMSO Affects Aβ1–40’s Conformation and Interactions with Aggregation Inhibitors. RSC Adv. 2015, 5, 69761–69764. 23. Cremades, N.; Cohen, S. I.; Deas, E.; Abramov, A. Y.; Chen, A. Y.; Orte, A.; Sandal, M.; Clarke, R. W.; Dunne, P.; Aprile, F. A.; et al. Direct Observation of the Interconversion of Normal and Toxic Forms of α-Synuclein. Cell 2012, 149, 1048–1059. 24. Kremer, F.; Schönhals, A. Eds. Broadband Dielectric Spectroscopy. Springer: Berlin (2003). 25. Levine, H. 3rd Thioflavine T Interaction with Synthetic Alzheimer’s Disease Beta-Amyloid Peptides: Detection of Amyloid Aggregation in Solution. Protein Sci. 1993, 2, 404–410. 26. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09; Gaussian, Inc.: Wallingford, CT (2009). 27. Zhao, Y.; Truhlar, D. The M06 Suite of Density Functionals for Main Group Thermochemistry, Thermochemical Kinetics, Noncovalent Interactions, Excited States, and Transition Elements: Two New Functionals and Systematic Testing of Four M06-Class Functionals and 12 Other Functionals. Theor. Chem. Acc. 2008, 120, 215–241. 28. Martín-Pintado, N.; Yahyaee-Anzahaee, M.; Deleavey, G. F.; Portella, G.; Orozco, M.; Damha, M. J.; González, C. Dramatic Effect of Furanose C2’ Substitution on Structure and Stability: Directing the Folding of the Human Telomeric Quadruplex with a Single Fluorine Atom. J. Am. Chem. Soc. 2013, 135, 5344–5347.

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29. Mompeán, M.; González, C.; Lomba, E.; Laurents, D. V. Combining Classical MD and QM Calculations to Elucidate Complex System Nucleation: a Twisted, Three-Stranded, Parallel β-Sheet Seeds Amyloid Fibril Conception. J. Phys. Chem. B 2014, 118, 7312–7316. 30. Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. A. A Consistent and Accurate Ab Initio Parametrization of Density Functional Dispersion Correction (DFT-D) for the 94 Elements H-Pu. J. Chem. Phys. 2010, 132, 154104.

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Figures

Figure 1. Structural organization of amyloid fibrils. (A) Monomers adopt an extended conformation (β-strand) and are noncovalent bound into β-sheets, perpendicularly stacked to the fibril axis. Backbone–backbone (bb–bb) H-bonds. (B) An additional network of side chain–side chain (sc–sc) H-bonds is found in containing at least one stacked Gln or Asn residue.

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Figure 2. HBC in amyloids. (A) Set up to study the interaction energies between different pair of strands. The color code corresponds to that in panels B and C. Although a parallel geometry is sketched, additional geometries were studied as shown in Supplementary Fig. S2, S5. (B) Interaction energy values for MVGGVV (triangles, aka Aβ35–40), GNNQQNY (open circles) and Aβ17–42 (open squares) are plotted within the same graph to allow a direct comparison of the three systems (note that each y-axis spans ~25 kcal/mol). (C) Interaction energies represented in separated plots to allow the reader appreciate the differences between polar and hydrophobic fibrils. Note that systems spanning more than three-stranded βsheets were not studied in Aβ17–42 due to the vast number of atoms involved.

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Figure 3. NBO analysis of the HBC. (A) The LP(N) à p-rich BD*(C=O) delocalization is a feature of CONH groups, and it is responsible for the basal-HBC in side chains (left) and backbone groups (right). (B) (Left) In Gln/Asn CONH-H groups, LP(N) is able to delocalize onto a sp-rich BD*(C=O) orbital, strengthening the HB interactions at bound strands (double red & green arrows), upon addition of an incoming monomer (note the circle at the newly formed H-bond). This interaction is not present in backbone CONH groups, since the N lone electron pair is delocalized onto the main chain (right). (C) Graphical representation of the two contributions to the HBC. The basal-HBC (purple, both panels) is ubiquitous to both the Q/N (left) and hydrophobic amyloids (right). Blue arrows in left panel emphasize the additional hyper-HBC term in GNNQQNY from a situation in which it would be absent (dotted lines). In the right panel, the opposite is shown: lack of hyper-HBC in MVGGVV and dotted lines indicate how the curves would look like in the presence of this contribution.

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Figure 4. Distinct behavior in Q/N and hydrophobic fibrils. (A) Relation of electrical conductivity to frequency (double log scale) of fibrils from GNNQQNY and Aβ in H2O (top) and DMSO (bottom). The DC conductivity component (σdc) obtained as the plateau at higher frequencies of these curves. GNNQQNY is shown in blue circles, and Aβ17-42 with red circles. The solvent is shown as black open circles (H2O) and filled black circles (DMSO). Fibrils dissolved in DMSO were immediately measured to avoid monomer contributions. (B) ThT assays in water and 100% DMSO-d6. Blue curves corresponds to GNNQQNY, whereas those in red represent data obtained on Aβ17–42. Continuous lines are used for parent samples in water, prior to DMSO treatment (control experiment). Denaturizing experiments in DMSO are indicated by dashed (1 day of incubation) and dotted lines (7 days of incubation). (C) 1D 1H NMR spectra of Aβ freshly dissolved in DMSO-d6 (bright red) and of Aβ fibrils incubated in DMSO-d6 (maroon). (D) 2D 1H TOCSY NMR spectra of GNNQQNY fibrils after incubation in DMSO-d6 (cyan), showing side chain amide H2N and Tyr Hδ-Hε crosspeaks. When transferred into D2O, all bb and sc amide protons are exchanged with solvent deuterons and only non-exchanging aromatic protons (Tyr Hδ-Hε) can be detected (blue).

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Complex System Assembly Underlies a Two ... - ACS Publications

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