Extraordinarily Stable Amyloid Fibrils Engineered from Structurally

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Extraordinarily Stable Amyloid Fibrils Engineered from Structurally Defined #-Solenoid Proteins Zeyu Peng, Maria delRefugio Peralta, and Michael D. Toney Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00364 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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Biochemistry

Extraordinarily Stable Amyloid Fibrils Engineered from Structurally Defined β-Solenoid Proteins

Zeyu Peng, Maria D.R. Peralta, Michael D. Toney*

Department of Chemistry, University of California Davis, 1 Shields Avenue, Davis, California 95616, United States

*Corresponding author: [email protected]

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ABSTRACT The self-assembly of biological molecules into ordered nanostructures is an attractive method for fabricating novel nanomaterials. Nucleic acid-based nanostructures suffer from limitations to functionalization and stability. Alternatively, protein-based nanostructures have advantageous chemical properties, but design facility lags that of nucleic acids. Structurally defined fibrils engineered from β-solenoid proteins (BSPs) form under mild conditions [ACS Nano (2015) 9(1):449-63], and are good candidates for novel nanomaterials because of the defined sequence-to-structure relationship and tunable properties. Here, the stability of two types of engineered fibrils was examined using CD, TEM, and electrophoresis. Both are stable to at least 90 °C, and one survives autoclaving. They are stable toward organic solvents, urea, and pH extremes. One is even stable in 2% SDS with heating. The fibrils show variable resistance to proteolytic digestion: one is resistant to trypsin, but chymotrypsin and proteinase K degrade both. These results show that BSPs have excellent potential for bottom-up design of rugged, functional, amyloid-based nanomaterials.

Key words: amyloid fibrils, β-solenoid proteins, bottom-up design, nanomaterials, thermal stability, chemical stability


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INTRODUCTION Biological self-assembly is an attractive method for fabricating ordered nanostructures.(1, 2) Structurally defined DNA-based nanostructures are well known, and allow ordered templating of nanoparticles. Unfortunately, they require nanoparticle functionalization to enable conjugation due to the limited chemistry of DNA bases (3-5), and have relatively low stability. Temperatures above ~60 °C (2), pH extremes (6), and denaturants readily disrupt them. Amyloid fibrils are a well known type of peptide/protein based nanostructure.(7, 8) They are defined as having a “cross-β” structure, in which β-strands run perpendicular to the long axis of the fibril.(9) Some forms of amyloid are soluble while others are not.(10, 11) Amyloids can be prepared from either proteins or peptides.(9, 12, 13) Examples include the self-assembly of a prion determinant from yeast (13) and the self-assembly of a diphenylalanine peptide that is the core recognition motif of Alzheimer’s β-amyloid peptide (12, 14, 15). The synthesis of amyloid fibrils generally requires harsh conditions for proteins that are not naturally amyloidogenic. Insulin amyloid fibrils form at very high temperatures (~100 °C),(16) while lysozyme amyloid fibrils form under acidic conditions.(17, 18) Once formed, amyloid fibrils are generally resistant to stress such as heat, organic solvents and proteases.(19-21) Detailed, systematic studies on the stability of amyloid fibrils are not numerous in the literature.(22-24) Amyloid fibrils formed from the α-spectrin SH3 domain were studied in detail by differential scanning calorimetry and were shown to form reversibly with a melting temperature of 75 °C and 1 mg/mL protein concentraion.(25) The pH dependence of insulin 3 ACS Paragon Plus Environment

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amyloid fibrils showed the importance of electrostatic interactions to dissociation.(26) Similarly, pH dependence was used to show that amyloid formation occurs maximally at the pI of proteins.(27) The N-terminal prion domain of Ure2p polymerizes to form amyloid fibrils that are stable to 100 °C.(19) Nanotubes synthesized from a diphenylalanine peptide are thermally and chemically stable; secondary structure is maintained to 90 °C, they survive autoclaving, and they are stable in a variety of organic solvents for 30 min.(20) Nanowires synthesized from the diphenylalanine peptide on solid substrates are stable up to 200 °C under dry conditions, at pH extremes, in polar and non-polar solvents for 12 h, and toward to proteinase K digestion.(21) For materials applications, facile synthesis of amyloid fibrils under mild conditions is needed, and self-assembly of engineered β-solenoid proteins (BSPs) provides it.(28) The peptide backbones of BSPs form solenoids of β-sheet with regular geometries (triangles, rectangles, etc.) (Figure S1). Herein, the thermal, chemical, and proteolytic stabilities of two BSP-based engineered amyloid fibrils are examined using circular dichroism spectroscopy (CD), transmission electron microscopy (TEM), thioflavin-T (ThT) fluorescence, and SDS-PAGE. The fibrils are derived from engineered antifreeze proteins (AFPs): one from the spruce budworm (SBAFP-m1),(28) and the other from the Rhagium inquisitor beetle (RiAFP-m9). RiAFP-m9 was engineered to self-assemble into micrometer-long fibrils using the same basic strategy employed previously for SBAFP-m1, which involves removal of capping structures and distortions at the ends of the monomer, regularization of the β-solenoid structure (i.e., removal of buldges and/or loops sometimes found at the corners of the rungs), and inclusion of salt bridges at the monomer interface.(28) SBAFP-m1 has a 4 ACS Paragon Plus Environment

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triangular cross-sectional shape and RiAFP-m9 has a rectangular one (Figure S2). Herein, it is shown that the engineered BSP fibrils are remarkably stable toward heat and chemical denaturants, but less so toward proteolysis. The high stability makes them attractive templates for engineering rugged new nanomaterials such as nanoscale-ordered scaffolds for inorganic nanoparticles, enzymes, antibodies, and peptides.


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Experimental Procedures Expression and purification of recombinant proteins. The RiAFP-m9 gene was synthesized by Thermo Fisher Scientific. The sequence of RiAFP-m9 is: MAHHHHHHSGSGSRAEARGEAMAEGHSRGCATSHANATGHADARSMSEGNAEAYTEAKGT AMATSEASGEARAQTNADGRAHSSSRTHGRADSTASAKGEAMAEGTSDGDAKSYASADGNAC AKSMSTGHADATTNAHGTAMADSNAIGEARAETRAEGRAESSSDTDGC The gene was cloned into pET-28a by Gibson assembly.(29, 30) Protein was expressed in E. coli BL21 (DE3) cells. Cultures were grown at 37 °C with shaking at 250 rpm until OD600 was 0.5 – 0.6. IPTG was added to a final concentration of 1 mM to induce expression. Four hours post induction, cells were collected by centrifugation (4,000 g for 20 min). The cells were resuspended in lysis buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM imidazole, pH 8) and were lysed by sonication. Soluble and insoluble proteins were separated by centrifugation (10,000 g for 30 min). RiAFP-m9 was expressed in the soluble fraction and purified by nickel affinity chromatography. SBAFP-m1 was expressed and purified as described previously.(28) Molecular models for monomers of both engineered proteins, in the form of pdb-formatted coordinate files, are included in Supplementary Materials.

Preparation of amyloid fibrils. The purified proteins were dialyzed into 10 mM sodium phosphate, pH 7.4. To promote fibril formation, 0.6 mg/mL of RiAFP-m9 was incubated at 37 °C for one week and 0.5 mg/mL of SBAFP-m1 were incubated at 37 °C for 10 days. Fibril formation is dependent on 6 ACS Paragon Plus Environment

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both temperature and protein concentration; higher temperature (tested up to ~50 °C) and higher protein concentration both increase the rate of fibril formation. The long incubation times used here ensure complete fibril formation under the conditions used.

Thermal stability of amyloid fibrils. The effect of heat on amyloid fibrils was studied by CD and TEM. Solutions of amyloid fibrils were incubated in a 1 mm path length CD cuvette. Spectra (190 nm to 300 nm) were recorded using an OLIS DSM 20 instrument at a scan rate proportional to high voltage, which was software controlled. This option reduces noise at wavelengths where light intensity at the PMT is reduced due to either lower lamp output or high sample absorbance. Each reported spectrum is an average of 3 scans. CD spectra of RiAFP-m9 and SBAFP-m1 fibrils were recorded at 37, 50, 60, 70, 80 and 90 °C; samples at the different temperatures were removed for TEM imaging. Specifically, the sample in the cell was held at a target temperature (starting at 37 °C) for 1 h. A 10 μL sample was removed and immediately loaded on a carbon coated copper grid for TEM, and the CD spectrum of the remaining fibrils was recorded. For TEM imaging, the protein sample was incubated on the grid for 2 min at room temperature to ensure the proteins attach to the grid surface. Excess liquid was removed by filter paper and the sample was stained with 2% uranyl acetate at room temperature for 30 s. Excess stain was removed by filter paper and the grid was dried at room temperature for 1-2 min. The specimen was imaged using a JOEL 1230 TEM with 100 kV of electron acceleration voltage. 7 ACS Paragon Plus Environment

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Protein fibrils were autoclaved at 121 °C, 17 psi, for 45 min, followed by TEM imaging. The effects of cold storage and freezing of fibrils were tested at 4 °C, -20 °C, and -80 °C for 24 h. Fibrils stored at -80 °C were rapidly frozen in liquid nitrogen before storing in the freezer. The frozen samples were thawed to room temperature before preparing grids for TEM imaging.

Chemical stability of amyloid fibrils. Solutions of protein fibrils were mixed with neat solvents (ethanol, methanol, 2-propanol and acetone) in a 1:1 ratio to give final concentration of 50% (v/v), and incubated at room temperature for 30 min. A 10 μL sample was loaded on a TEM grid for imaging, and the rest of the sample was kept at room temperature for 24 h. Stability toward denaturants was tested by dialyzing fibrils against 10 mM sodium phosphate containing either 8 M urea, or 2 M or 6 M guanidine hydrochloride, pH 7.4 for 24 h. The effect of SDS was tested on protein monomers and fibrils. Protein solutions (0.6 mg/mL of RiAFP-m9; 0.5 mg/mL of SBAFP-m1) were mixed 1:1 with 2× SDS-PAGE loading buffer containing 4% SDS and heated at 95 °C for 5 min. Both monomers and fibrils were subjected to SDS-PAGE. Additionally, 4% SDS in 10 mM sodium phosphate, pH 7.4 was mixed 1:1 with fibrils and the solution heated at 95 °C for 5 min. Fibrils were dialyzed against acidic (10 mM sodium phosphate + HCl, pH 2) or basic (10 mM sodium phosphate + NaOH, pH 11) solutions for 24 h to examine the pH dependence of stability. The stability of BSP fibrils in the presence of high salt was tested by dialyzing fibrils 8 ACS Paragon Plus Environment

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against 10 mM sodium phosphate (pH. 7.4) containing 2.5 M NaCl for 24 h. All of the above samples were imaged by TEM.

ThT fluorescence assays. ThT stock solutions were prepared by dissolving ~2 mg of ThT in 2 mL of PBS, pH 7.4 and filtering through a 0.22 µm filter. Stock concentrations were determined using an extinction coefficient of 26,620 M-1cm-1 at 416 nm. A 500 µM solution was prepared from the stock solution. In the assay, protein was added a final concentration of 5 µM and ThT to a final concentration of 10 µM in PBS, pH 7.4, and the solution was incubated at room temperature for 10 min. The emission spectrum was then recorded from 465 nm to 600 nm, with excitation at 450 nm.

X-Ray powder diffraction. The RiAFP-m9 sample (~30 mg/mL) in PBS was dried under vacuum at room temperature in a speedvac, then ground into a fine powder with a plastic pipette tip before analysis. Data were collected using a Bruker D8 Advance with Copper Kα radiation (λ = 1.54060 Å). The tube voltage was 40 kV, the current was 25 mA, and scans from 2θ = 5 to 120 deg were measured with a step of 0.01 deg at 1s per step.

Proteolytic stability of amyloid fibrils. Trypsin (0.2 mg/mL) was prepared in 10 mM sodium phosphate, pH 7.4. The protein fibrils were incubated with trypsin (20:1 w/w) at 37 °C for 24 h. Chymotrypsin (2 mg/mL) 9 ACS Paragon Plus Environment

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was prepared in 100 mM Tris-HCl, 10 mM CaCl2, pH 7.8. The protein fibrils were dialyzed into the same buffer, and incubated with chymotrypsin (60:1 w/w) at 30 °C for 24 h. Proteinase K (20 mg/mL) was prepared in 50 mM Tris-HCl, 1 mM CaCl2, pH 8. The protein fibrils were dialyzed into the same buffer. RiAFP-m9 fibrils (0.6 mg/mL) were incubated with proteinase K (0.05 mg/mL, 0.1 mg/mL and 1 mg/mL) at 37 °C for 2 h. SBAFP-m1 fibrils (0.5 mg/mL) were incubated with proteinase K (0.02 mg/mL) at 37 °C for 4 h. For analyses, a 10 μL RiAFP-m9 sample was loaded on a TEM grid for imaging and SBAFP-m1 fibrils before and after digestion were analyzed by SDS-PAGE and TEM. Bovine serum albumin (BSA) digested by trypsin, chymotrypsin, and proteinase K served as positive controls (Figure S6). BSA was treated with each enzyme under the same conditions employed for RiAFP-m9 fibrils. The samples were subjected to SDS-PAGE to verify proteolytic digestion.


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RESULTS Thermal stability of RiAFP-m9 and SBAFP-m1 fibrils. The thermal stability of RiAFP-m9 and SBAFP-m1 fibrils was studied with CD and TEM. Fibril samples were heated stepwise. After holding the sample at the target temperature for 1 h, an aliquot was loaded on a TEM grid for imaging, and the CD spectrum of the sample was taken. Figure 1 shows CD spectra of RiAFP-m9 and SBAFP-m1 fibrils in the temperature range of 37 – 90 °C. The RiAFP-m9 spectra indicate stable β-sheet throughout the temperature range studied (Figure 1A). Even at 90 °C, there is no indication of loss of β-sheet structure as judged by the stable negative peak at ~218 nm. The SBAFP-m1 spectra indicate stable β-sheet structure up to ~80 °C, but there appears to be secondary structure changes that occur near 90 °C (Figure 1B). The β-sheet content of RiAFP-m9 polymerized at 37 °C but otherwise unheated was additionally confirmed by ThT fluorescence (Fig. S8) and powder X-ray diffraction (Fig. S9).

Fig. 1. CD spectra of RiAFP-m9 and SBAFP-m1 fibrils. A) RiAFP-m9 fibrils at 37, 50, 60, 70, 80 and 90 °C. B) SBAFP-m1 fibrils at 37, 50, 60, 70, 80 and 90 °C. TEM results correlate well with those from CD. Figures 2 and 3 show TEM images of 11 ACS Paragon Plus Environment

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RiAFP-m9 and SBAFP-m1 fibrils at various temperatures. At 90 °C, both RiAFP-m9 and SBAFP-m1 fibrils are present. The limit of thermal stability for both RiAFP-m9 and SBAFP-m1 fibrils was tested by autoclaving them under 17 psi steam pressure at 121 °C for 45 min. After autoclaving, fibrils were present in the RiAFP-m9 sample (Figure 2G and 2H), but were absent in the SBAFP-m1 sample. The SBAFP-m1 sample after autoclaving gave a ThT fluorescence signal that was only slightly above background compared to protein before autoclaving.

Fig. 2. TEM images of RiAFP-m9 fibrils at different temperatures. A) 37 °C. B) 50 °C. C) 60 °C. D) 70 °C. E) 80 °C. F) 90 °C. G & H) Autoclaved fibrils (121 °C, 17 psi, 45 min). Scale bar: 200 nm. The cold stability of the fibrils was tested. Storage of the fibrils at 4 °C for 24 h has no obvious effect on their structure (Figure S3). Freezing at -20 °C and -80 °C causes RiAFP-m9 fibrils to fragment, while SBAFP-m1 fibrils remain long but appear to associate into thicker fibrils (Figure S3).


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Fig. 3. TEM images of SBAFP-m1 fibrils at different temperatures. A) 37 °C. B) 50 °C. C) 60 °C. D) 70 °C. E) 80 °C. F) 90 °C. Scale bar: 200 nm.

Chemical stability of RiAFP-m9 and SBAFP-m1 fibrils. Fibrils were mixed 1:1 with organic solvents and incubated at room temperature for 30 min and 24 h. Ethanol, methanol, 2-propanol and acetone (final concentration of 50% v/v) show no dramatic disruptive effect on either RiAFP-m9 or SBAFP-m1 fibrils (Figures 4 and 5). Incubation with 8 M urea for 24 h has no apparent effect on either RiAFP-m9 or SBAFP-m1 fibrils (Figure 6). Fibril stability was also tested in 2 M guanidine hydrochloride; SBAFP-m1 fibrils remain intact (Figure S4), but RiAFP-m9 fibrils dissociate. Both types of fibrils dissociate in 6 M guanidine hydrochloride. The ThT fluorescence spectrum of RiAFP-m9 incubated in 2 M guanidine hydrochloride was essentially identical to that for RiAFP-m9 kept at 4 °C (Fig. S8), consistent with the fibrils dissociating into monomers. 13 ACS Paragon Plus Environment

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Fig. 4. TEM images of RiAFP-m9 fibrils in organic solvents (50% v/v). Fibrils were incubated with the indicated solvent for 30 min and 24 h. Scale bar: 200 nm.

Fig. 5. TEM images of SBAFP-m1 fibrils in organic solvents (50% v/v). Fibrils were incubated with the indicated solvent for 30 min and 24 h. Scale bar: 200 nm. RiAFP-m9 does not appear as a monomer on SDS-PAGE after heating at 95 °C for 5 min in loading buffer containing SDS (Figure 7A). TEM images confirm that RiAFP-m9 fibrils remain intact after heating at 95 °C for 5 min in 2% SDS (Figure 7B) with possible


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morphological changes. In contrast, SBAFP-m1 appears as a monomer on SDS-PAGE after heating at 95 °C for 5 min in SDS loading buffer (Figure 9B and 9C), and TEM images (not shown) indicate no SBAFP-m1 fibrils are present. ThT assays on SBAFP-m1 heated in SDS were not possible due to the increase in background ThT fluorescence in the presence of SDS.

Fig. 6. TEM images of fibrils in 8 M urea. Fibrils were incubated in 8M urea for 24 h. A) RiAFP-m9 fibrils. B) SBAFP-m1 fibrils. Scale bar: 200 nm.

Fig. 7. Analysis of RiAFP-m9 fibrils after heating in 2% SDS at 95 °C for 5 min. A) SDS-PAGE of RiAFP-m9 monomers and fibrils. Very little RiAFP-m9 monomer enters the SDS-PAGE gel after heating in SDS. The majority of the protein in the sample does not enter the gel. B) TEM image of the fibrils after heating in 2% SDS. Scale bar: 200 nm. Fibrils of both BSPs incubated at pH 2 and pH 11 for 24 h remain intact (Figure 8).


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SBAFP-m1 fibrils incubated in buffer containing 2.5 M NaCl are stable and appear to exhibit some lateral assembly (Figure S5), which is consistent with previous study showing that fibrils align in the presence of salt.(31) On the other hand, RiAFP-m9 fibrils dissociate in 2.5 M NaCl as determined by TEM and ThT. Since RiAFP-m9 fibrils are stable against denaturants such as urea and SDS, NaCl induced dissociation of RiAFP-m9 fibrils into monomers suggests that the dissociative effect of 2 M guanidine hydrochloride on RiAFP-m9 might be largely due to ionic strength rather than its chaotropic properties.

Fig. 8. TEM images of fibrils under acidic and basic conditions. The effects of pH on fibrils were measured by TEM. Fibrils were observed after 24 h in pH 2 and pH 11 solutions. Scale bar: 200 nm.


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Proteolytic stability of RiAFP-m9 and SBAFP-m1 fibrils. Fibril resistance to enzymatic proteolysis was examined. RiAFP-m9 fibril stability was examined using TEM only since the fibrils are stable toward boiling in SDS-PAGE loading buffer. After incubation with trypsin (20:1 w/w fibril-to-protease, 37 °C, 24 h), RiAFP-m9 fibrils are abundant in TEM images (Figure 9A), whereas no fibrils were found by TEM after treatment with chymotrypsin (60:1 w/w fibril-to-protease, 30 °C, 24 h) or proteinase K (0.05 - 1 mg/mL enzyme, 37 °C, 2 h). SDS-PAGE analysis of SBAFP-m1 fibrils shows that trypsin and chymotrypsin both digest them (Figure 9B). Proteinase K (0.02 mg/mL enzyme, 37 °C, 4 h) also hydrolyzes SBAFP-m1fibrils (Figure 9C).

Fig. 9. Amyloid fibrils treated with proteases. A) TEM image of RiAFP-m9 fibrils treated with trypsin (20:1 w/w fibril-to-protease, 37 °C, 24 h). Scale bar: 200 nm. RiAFP-m9 monomers are not observed on SDS-PAGE gels due to the stability of the fibrils toward boiling in loading buffer. Therefore, TEM was used to determine the presence or absence of fibrils. B) SDS-PAGE of SBAFP-m1 fibrils before and after trypsin (20:1 w/w fibril-to-protease, 37 °C, 24 h) and chymotrypsin (60:1 w/w fibril-to-protease, 30 °C, 24 h) treatments. C) SDS-PAGE of SBAFP-m1 fibrils before and after proteinase K (0.02 mg/mL, 37 °C, 4 h) treatment. TEM images confirmed the absence of fibrils in the SBAFP-m1 samples after treatment. 17 ACS Paragon Plus Environment

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DISCUSSION Here, TEM is used to determine the presence and structure of BSP fibrils. It is known that sample preparation or contaminants in the sample can introduce TEM artifacts such as interfibrillar bonding,(32) shrinkage of fibrils,(32) fibril-like structures in water and PBS(33) and the rouleau artifacts of lipoproteins.(34, 35) Cellular TEM artifacts include distorted membranes,(36) cell shrinkage,(37) collapse of cell structure,(37) and redistribution and denaturation of molecular components.(37) The concern here is whether the fibrils in the TEM images are present in solution or artifactual. To address this issue, we imaged SBAFP-m1 monomers before the extended 37 °C polymerization step under the same conditions used here and no fibrils were observed (Figure S7). This shows that the drying of the sample on the TEM grid and subsequent staining does not induce rapid polymerization. Ultra-sonication is known to fragment long amyloid fibrils (38). Ultra-sonication of both RiAFP-m9 and SBAFP-m1 shortens both types of fibrils to ~100 nm (manuscript submitted) and TEM sample preparation does not induce them to repolymerize toward the original micron-long fibrils. Additionally, RiAFP-m9 treated with high salt shows no fibrils, and freezing it yielded fragmented fibrils even after thawing the sample at room temperature. SBAFP-m1 that was autoclaved or treated with SDS shows no fibrils. These observations support the conclusion that the species observed here by TEM represent the state of the fibrils in the solution from which the samples were taken. Amyloid fibrils form from natural fibril-forming proteins (13), or from various other proteins under harsh incubation conditions (16, 18). The approach used here is unique in that it employs engineering structurally defined, intrinsically non-amyloidogenic BSPs to 18 ACS Paragon Plus Environment

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enable their self-assembly into amyloid fibrils under benign conditions. It is promising that the engineered BSP amyloid fibrils share the intrinsic stability of other amyloid fibrils.(19-21) From a materials point of view, this ruggedness is highly advantageous. The stabilities of both RiAFP-m9 and SBAFP-m1 fibrils are summarized in Table 1. Both are stable toward heat. CD spectra show that the β-sheet structure of RiAFP-m9 fibrils is stable to at least 90 °C. TEM shows that RiAFP-m9 fibrils are present even after autoclaving. CD analysis shows that SBAFP-m1 fibrils retain β-sheet structure up to ~80 °C, while TEM shows SBAFP-m1 fibrils remain at 90 °C but not after autoclaving. Table 1. Stability of SBAFP-m1 and RiAFP-m9 fibrils.1 Conditions RiAFP-m9 SBAFP-m1 Heat (90 °C) √ √ Autoclaving √ × Freezing (-20 °C or -80 °C) * √ Organic solvents √ √ 8 M urea √ √ 2% SDS, 95 °C for 5 min √ × 2 M guanidine hydrochloride × √ Extreme pH (2 or 11) √ √ √ High salt (2.5 M NaCl) × Trypsin √ × Chymotrypsin × × Proteinase K × × 1 √, Fibrils present. ×, Fibrils absent. *, Fibrils fragmented. Denaturation and/or precipitation commonly occurs when proteins are exposed to organic solvents, urea, SDS, or extremes of pH, but the two BSP-derived amyloid fibrils tested here are resistant to them. Both RiAFP-m9 and SBAFP-m1 fibrils survive exposure to ethanol, methanol, 2-propanol, and acetone. Surprisingly, RiAFP-m9 fibrils are stable even after treatment with 2% SDS at 95 °C for 5 min. The results of proteolysis studies are varied. RiAFP-m9 has 11 arginine and 4 lysine


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residues, yet it remains intact after incubation with trypsin. However, chymotrypsin hydrolyzes RiAFP-m9 fibrils even though two tyrosines are the only potential cleavage sites. Proteinase K, which has a broad specificity,(39) digests both RiAFP-m9 and SBAFP-m1 fibrils. The stability of the BSP amyloid fibrils presented here is extraordinary compared to average globular proteins and many amyloid fibrils, raising the obvious question of “why”. Two possible sources of stability are discussed here. The first is the dense hydrogen bonding pattern of both of the proteins (Figure 10A and 10B). Each amide bond of the polypeptide backbone participates in two hydrogen bonds: one as a donor and one as an acceptor. This structure makes the hydrogen bonds cooperative, which increases their strength. The average globular protein has 1.1 hydrogen bonds per residue.(40) The two proteins studied here have more than double this number: RiAFP-m9 has an average of 2.5 hydrogen bonds per residue while SBAFP-m1 has 2.6. Thus, one expects the intrinsic tertiary structures of the BSPs to be substantially more stable than the average globular proteins on a per residue basis. A second source of the high fibril stability is likely related to the Zimm-Bragg model of helix-coil transitions, which takes the cooperativity of each segment into account.(23, 41) In this model, addition of a helical section to an already helical core (propagation) is much more favorable than de novo formation of a helical section (nucleation). In fibrils composed of short peptides, with each peptide forming a rung of the cross β-sheet ladder, breakage of a rung of hydrogen bonds perpendicular to the fibril axis necessarily leads to two separate molecules with independent diffusive freedom. Entropically, this poses a substantial penalty for reformation of the original extended fibril. The fibrils studied here, on the other hand, 20 ACS Paragon Plus Environment

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are statistically most likely to absorb energy in a rung(s) of the solenoid not at the monomer-monomer interface; there eight rungs of the solenoid per interface in RiAFP-m9, and twelve rungs per interface in SBAFP-m1. If a non-interface rung is disrupted, the diffusive barrier to reforming the intact fibril is low due to the covalent linkage between the proximal β-helical segments. This leads to rapid reformation of β-helical structure in the disrupted segment because helical nuclei are present at both ends and only propagation need occur for the disrupted segment between them. Combined, the large number of hydrogen bonds per residue and the rapid reformation of β-helical structure by propagation once the cross-β structure is disrupted are likely the central factors governing the extreme fibril stability observed here. The stability of RiAFP-m9 is apparently greater than that of SBAFP-m1 as judged by its higher thermal stability and the resistance to denaturation by SDS. In light of the discussion above, these differences may originate in the different number of hydrogen bonds per turn of the β-solenoid structure, as well as differences in the monomer-monomer interface structures. RiAFP-m9 has 20 residues per turn while SBAFP-m1 has only 15. The monomer-monomer interface structures are shown in Figure 10C and 10D. SBAFP-m1 has, in addition to the salt bridge formed by the chain termini, 2 additional salt bridges engineered into the interface, while RiAFP-m9 has 6 additional salt bridges plus a disulfide bond across the interface. For SBAFP-m1, a total of 15 hydrogen bonds and 3 salt bridges must be broken at the monomer-monomer interface to cause fibril fragmentation, while for RiAFP-m9 a total of 20 hydrogen bonds, 7 salt bridges, and one disulfide bond must be broken. RiAFP-m9 has an energetic advantage of 5 hydrogen bonds, 4 salt bridges, and one disulfide bond holding 21 ACS Paragon Plus Environment

Biochemistry

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its monomer-monomer interface together over that of SBAFP-m1. An underappreciated fact about water is that its dielectric constant decreases significantly with increasing temperature. For example, it is 78 at 25 °C and drops to 56 at 100 °C.(42) This dramatically increases the strength of ionic interactions at higher temperatures, enhancing the stability advantage of the RiAFP-m9 interface. Combined, these numbers provide a credible explanation for the higher stability of RiAFP-m9 over SBAFP-m1 fibrils. Several advantages of using fibrils engineered from BSPs are evident. The engineering of linear fibrils is simple and robust. To date, three (including RiAFP-m9) published successes(28) and other unpublished ones from this laboratory show that the general procedure of regularizing the β-solenoid backbone and creating an intimate monomer-monomer interface suffices. As demonstrated herein, the fibrils formed by this general procedure are extraordinarily robust toward stress. Importantly, the chemical diversity of amino acid side chains allows much simpler derivatization compared to the limited chemistry of the nucleic acid bases. This allows a large number of known protein chemical modifications to be applied wholesale.(43-45) Moreover, the addition of peptide tags (e.g., RiAFP-m9 has a 6×His tag on the N-terminus) to the modified BSPs has no effect on their self-assembly into fibrils. This bodes well for creating material-binding fibrils simply by attaching known material-specific peptides.(46-51) The next challenge is to create higher dimensional materials from one dimensional fibrils. This will involve, for example, engineering interfaces between fibrils to create two-dimensional sheets. The rapid development of protein design software such as Rosetta(52) and the successes achieved in such endeavors(8) predict a bright future in this direction. 22 ACS Paragon Plus Environment

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Fig. 10. Models of the proteins studied. The carbons of the different monomers are colored pink and grey for clarity. Hydrogen bonds are shown as yellow dashed lines. Orthogonal views of (A) SBAFP-m1 and (B) RiAFP-m9. Charged residues of (C) SBAFP-m1 and (D) RiAFP-m9 are shown in ball-and-stick. The locations of the interfaces are indicated by the arrows. The interfacial disulfide bond in RiAFP-m9 is labeled. 23 ACS Paragon Plus Environment

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CONCLUSIONS Amyloid fibrils are readily engineered from BSPs, and these fibrils show extraordinary stability toward heat, extremes of pH, organic solvents, and other denaturants. The diverse side-chain chemical functionality of proteins combined with the flat faces of BSPs should enable the development of novel, complex, highly structured, protein-based nanoscale scaffolds using available software. These scaffolds may find application in a variety of fields such as delivery of biological therapeutics at wound sites, ordering of inorganic nanoparticles for thermoelectrics and photovoltaics, and ordering enzymes into metabolic pathways.


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ACKNOWLEDGEMENTS The authors would like to thank Dr. Fei Guo from Electron Microscopy Imaging Facility, Department of Molecular and Cellular Biology, UC Davis, for training and assistance with TEM.

FUNDING SOURCES This work was supported by Research Investments in the Sciences and Engineering (RISE) program from the UC Davis Office of Research.

SUPPLEMENTARY MATERIALS Supplementary materials are available online. This includes molecular models, TEM images, SDS-PAGE results, and pdb formatted structure files for RiAFP-m9 and SBAFP-m1 models.


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Extraordinarily Stable Amyloid Fibrils Engineered from Structurally

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