Structural and Mechanical Properties of Amyloid Beta Fibrils: A

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Structural and Mechanical Properties of Amyloid Beta Fibrils: A Combined Experimental and Theoretical Approach Thomas J. Paul,† Zachary Hoffmann,† Congzhou Wang,§ Maruda Shanmugasundaram,‡ Jason DeJoannis,Δ Alexander Shekhtman,*,‡ Igor K. Lednev,*,‡ Vamsi K. Yadavalli,*,§ and Rajeev Prabhakar*,† †

Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia 23284, United States ‡ Department of Chemistry, University at Albany, State University of New York, Albany, New York 12222, United States Δ Dassault Systèmes BIOVIA, San Deigo, California 92121, United States §

S Supporting Information *

ABSTRACT: In this combined experimental (deep ultraviolet resonance Raman (DUVRR) spectroscopy and atomic force microscopy (AFM)) and theoretical (molecular dynamics (MD) simulations and stress−strain (SS)) study, the structural and mechanical properties of amyloid beta (Aβ40) fibrils have been investigated. The DUVRR spectroscopy and AFM experiments confirmed the formation of linear, unbranched and β-sheet rich fibrils. The fibrils (Aβ40)n, formed using n monomers, were equilibrated using all-atom MD simulations. The structural properties such as β-sheet character, twist, interstrand distance, and periodicity of these fibrils were found to be in agreement with experimental measurements. Furthermore, Young’s modulus (Y) = 4.2 GPa computed using SS calculations was supported by measured values of 1.79 ± 0.41 and 3.2 ± 0.8 GPa provided by two separate AFM experiments. These results revealed size dependence of structural and material properties of amyloid fibrils and show the utility of such combined experimental and theoretical studies in the design of precisely engineered biomaterials.

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iomaterials encompass various facets of medicine,1 biology,2 chemistry,3,4 and materials science.5 Their applications include scaffolds for cell culture6−10 and catalytic reactions,11−15 devices,16−23 and bioimplants.6,24,25 However, the majority of materials used in these applications consist of classical nonbiological polymeric molecules.26 Biological materials possess lower immunogenic and inflammatory potential than nonbiological polymers27 and, thus, would be better suited for fabrication of artificial body parts and tissue scaffolds in the fields of tissue engineering and regenerative medicine.28 Additionally, these materials can be used as building blocks for electronic devices and nanowires.29,30 Amyloid beta (Aβ) peptides are promising biomolecules that are capable of forming a variety of materials under diverse conditions.3,4,31−34 Their stability, accurate self-assembly,35 and easy functionalization6 provide an excellent set of material properties that can be exploited for the aforementioned applications.28,36 Driven by intermolecular forces such as hydrogen bonds, electrostatic and hydrophobic interactions, and π−π stacking, they can self-assemble molecule by molecule to produce supramolecular architectures (fibrils). This process proceeds through the formation of a natively unfolded intermediate to produce energetically stable, highly ordered, and β-sheet-rich fibrils.37 The fibrils possess characteristic morphologies (hollow cylinders, twisted, and flat ribbons) ∼ © XXXX American Chemical Society

100 Å in diameter and have variable lengths up to several micrometers.3,38−42 The fibrils formed by small fragments of Aβ peptides possess high mechanical strength, elasticity, thermochemical stability, and self-healing.4,27,43−45 These properties compare very favorably to most proteinaceous and nonproteinaceous materials.46 They are most likely related to their macromolecular nature and in particular, to the physical and chemical constraints imposed by the individual amino acid residues. However, due to their heterogeneity, high-resolution molecular structures of low molecular weight Aβ amyloid oligomers cannot be easily determined because they are noncrystalline solid materials which are not amenable to X-ray crystallography and liquid state NMR.47−53 Additionally, due to the fast rate of aggregation, structural determination of the early aggregates by using these experimental techniques is extremely difficult. Despite the availability of a sizable amount of data, there are no systematic studies to elucidate the roles of amino acid sequence and structure pertaining to the fundamental material properties such as great strength, sturdiness, and elasticity. Here we have combined deep ultraviolet resonance Raman (DUVRR) spectroscopy and atomic force microscopy (AFM) Received: May 17, 2016 Accepted: July 7, 2016

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Letter

The Journal of Physical Chemistry Letters techniques with molecular dynamics (MD) simulations and stress−strain (SS) calculations to derive a fundamental understanding of the sequence-structure-material properties relationship of these materials. In particular, structural and mechanical properties such as secondary structure and Young’s modulus (Y) of the Aβ40 fibrils provided by the DUVRR spectroscopy and AFM experiments are compared with the corresponding properties of (Aβ40)n fibrils, for n = 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 80, and 120, where n is the number of monomers, derived from MD simulations and SS calculations. These results will elucidate size dependence of structural and mechanical properties of the Aβ40 fibrils and help the development of design rules for the accurate modeling of biomaterials. Structural Properties of Aβ40 Fibrils. The AFM imaging of Aβ40 fibrils revealed linear, unbranched structures, which are typical for fibrillary aggregates (Figure 1a). The fibril lengths

Specifically, a narrow and intense Amide I peak centered at 1675 cm−1 is indicative of a well-organized cross-β core structure of amyloid fibrils.56 The Amide I vibration consisting mainly of CO stretching and a small contribution from outof-phase C−N stretching is known to be sensitive to the peptide’s secondary structure.57 The details of AFM and DUVRR experiments are provided in the Supporting Information (SI). The structures of a wide range of (Aβ40)n fibrils, for n = 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 80, and 120 where n is the number of monomers, were equilibrated using all-atom 50− 100 ns MD simulations. They were performed using the GROMACS 4.5.6 software58,59 utilizing the GROMOS96 53A6 force field59 in explicit aqueous solution. The details of simulations are provided in the SI. Amyloid fibrils were essentially grown in-silico, starting with a Aβ40 fibril structure that was generously provided by Robert Tycko using a solidstate nuclear magnetic resonance (NMR) method (PDB ID: 2LMN).37 The root-mean-square-deviations (rmsd) confirmed that the structures were equilibrated during the simulations. The accuracy of the simulated structures was further validated by comparing them with experimental DUVVR and NMR data such as secondary structure analysis, periodicity, and interstrand twist and angle.60 In Aβ40 fibrils, side-chains emanating from the two separate monomer sheets were found to be tightly interdigitated like the teeth of a zipper by intermolecular forces such as hydrogen bonding, π−π interactions, and CH−π interactions.61 Additionally, there was an absence of water between the β-sheets, therefore this motif has been termed the “dry steric zipper.”61 Steric zippers were formed from selfcomplementary amino acid sequences, in which their sidechains could mutually interdigitate.61 The fibrils were found to be mostly β-sheet (88.0%) in character with small unordered sections (12.0%) at the beginning of each monomer sequence (Table 1). The formation of the β-sheet rich structures was supported by the measured DUVRR data. The larger fibril (n > 30) structures began to twist in order to gain structural stability and minimize repulsion (Figure 2). Smaller fibrils (100 monomers) approach a more realistic value when compared with traditional AFM stress experiments. 2761

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The Journal of Physical Chemistry Letters

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Table 2. Computed Values of Young’s Modulus (Y) as a Function of Aβ40 Monomers monomer units

Young’s modulus (GPa)

5 6 7 8 9 10 20 30 40 50 60 80 120

38.0 36.2 37.3 54.1 44.5 51.2 54.3 26.0 33.9 19.8 11.4 6.1 4.5

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Tel: 305-284-9372, Fax: 305-2844571. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation under Grant No. CHE-1152752 (I.K.L.). Financial support from the James and Esther King Biomedical Research Program of the Florida State Health Department (DOH grant number 08KN-11) to R.P. is gratefully acknowledged. Computational resources from the Center for Computational Science at the University of Miami are greatly appreciated.

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REFERENCES

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In this study, we have combined complementary experimental (DUVRR and AFM) and theoretical (MD simulations and SS calculations) techniques to investigate the structural and mechanical properties of Aβ40 fibrils. The AFM experiments showed the formation of linear and unbranched Aβ40 fibrils with varying length (a few hundred nanometers to a few microns) and thickness (3−5 nm). The DUVRR spectrum provided a high relative intensity of the Amide I peak at 1675 cm−1 that was characteristic of β-sheet rich fibrils. The MD equilibrated fibrils formed using 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 80, and 120 monomers were also dominated by β-sheets (88.0%) and formed through a zipper created by selfcomplementary amino acid residues. The other structural properties (twist, interstrand distance and periodicity) of these fibrils were also in agreement with experimental measurements. The AFM experiments provided the values of compressive Y of 1.79 ± 0.41 GPa (sample size = 20 fibrils) in air (dry) condition. The SS calculations on the small equilibrated (Aβ40)5−(Aβ40)20 fibrils provided an average Y value of 45.0 ± 7.5 GPa that was significantly higher than the measured value. However, the larger structures (Aβ40)40−(Aβ40)120, exhibited a monoexponential decay and produced a high correlation value of 0.99, which suggested the existence of a scaling law. The monoexponential decay using this law provided Y = 4.206 GPa that can be associated with any structure greater than 200 units long. This value is in excellent agreement with AFM nanoindention experimental value of 1.8 ± 0.41 GPa. The results reported in this study will advance our efforts to understand sequence-structure-material properties relationship of biomaterials and to develop “design rules” for their computational modeling.

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Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b01066. Complete references for refs 6, 25, 50, and 53 from the main text as well as further details on experimental and computational procedures pertaining to preparation of Aβ40 fibrils samples, deep ultraviolet resonance Raman (DUVRR) spectroscopy, AFM imaging, nanoindentation approach, computational modeling, molecular dynamics simulations, geometrical parameters, and stress−strain calculations (PDF) 2762

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