Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides
© Scotter et al; licensee BioMed Central Ltd. 2007
Received: 15 March 2007
Accepted: 01 October 2007
Published: 01 October 2007
A right-handed, calcium-dependent β-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca2+. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide.
Initial analysis of the two polypeptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by right angle light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains the metal ion-dependency. The model is supported by the capping of one of the polypeptides with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments.
Metal ion-dependent, reversible protein filament formation is demonstrated for two designed polypeptides. The polypeptides form filaments that are approximately 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the "decoration" of a protein filament with various functional moieties.
It has also previously been put forward that parallel β-strands and β-helical or β-roll repeating motifs are found in many amyloid filaments and fibrils . To date, the β-roll model of HET-s fibrils from Podospora anserina  is the most plausible β-roll-like model of an amyloid filament and has been supported by recent electron miscroscopy studies  in addition to mutational analysis of HET-s derived protein sequences.
β-roll domains have been found in a number of proteins, often as a subdomain that binds divalent metal ions (Ca2+), as in the "zincin" family of metalloproteases [6–8]. including alkaline protease from Pseudomonas aeruginosa, see Figure 1 to indicate the position of the β-roll domain within the protease. This β-roll is composed of glycine- and aspartate-rich nonapeptide repeats with a GGXG(X/D)DXUX consensus sequence (where G = glycine, X = any amino acid, D = aspartate, and U = a large hydrophobic residue such as Leu, Ile, Val, Phe, Tyr) .
Similar nonapeptide repeats are present in the C-terminal regions of the 100 – 200 kDa repeat-in-toxin (RTX) family of cytotoxic and hemolytic toxins from Gram negative bacteria [9–15] and the I.3 family of lipases [16, 17]. In addition, a right-handed β-roll domain containing nonapeptide repeats has been structurally characterized in the R-module of the AlgE4 C-5 epimerase from Azotobacter vinelandii by nuclear magnetic resonance (NMR) . Structural and experimental data suggest that calcium ions can be bound within the turns of the R-module β-roll domain to stabilize the overall protein.
The RTX metal-binding nonapeptide repeat has been the subject of two previous studies that exploit the change in conformation of the motif upon calcium binding. A study by Lilie et al. used a synthetic 75-residue polypeptide (NH2-WLS [GGSGNDNLS]8-COOH) as a minimized model of the β-roll domains from RTX toxins . The synthetic β-roll irreversibly bound calcium in the millimolar range and showed a change in conformation in the presence of 100 mM CaCl2 and 25% PEG 8000 as monitored by far-UV circular dichroism (CD). The minimized β-roll behaved similarly to the β-roll regions of B. pertusis adenylate cyclase toxin and E. coli hemolysin. The early success of minimizing the β-roll domain was followed by a study using the GGXGXDXUX nonapeptide repeat β-roll motif from Serratia marcescens serralysin as a calcium switching "molecular spring" fused between two 6-phospho-β-galactosidase (PGAL) monomers . In the presence of calcium, the β-roll formed a 2 nm long rod linking the two PGAL units, similar to a molecular dumbbell when viewed by electron microscopy (EM). Upon addition of ethylenediaminetetraacetic acid (EDTA) the β-roll adopted an extended conformation and the two PGAL units became more distant from one another as demonstrated by EM.
BRD 1 was designed as a 34-amino-acid minimized β-roll motif containing three β-strands and two calcium-binding sites, see Figure 2B and 2C. BRD1 was intended to be stabilized by the coordination of two metal ions and the introduction of a pair of stacked tryptophan residues (W16 and W34), which have previously been shown to stabilize short, soluble, β-hairpin proteins . Residues 9 (I to K) and 27 (Y to E) of BRD1 were altered from the alkaline protease sequence to introduce a potential salt bridge between β-strands. BRD2 is an elongated version of BRD1 (50 amino acids) that is comprised of five β-strands with four calcium-binding sites, and also includes a stacked pair of tryptophan residues (W27 and W45) as shown in Figure 2D and 2E. Once again charged residues were introduced to the sequence (L to Q at position 16 and I to K at position 34) to promote stability and solubility. The N-terminal extension contains a modified nonapeptide repeat with asparagine side chains to potentially bind calcium and a glutamic acid residue to add further charged character to the polypeptide. Several glycine residues were replaced with alanine to limit the flexibility of the BRD2 polypeptide. The folded BRD2 motif is approximately 30 Å wide, 25 Å deep and 15 Å high.
The BRD polypeptides were originally designed to fold upon the addition of calcium and form soluble monomeric β-rolls that could be used as scaffolds for the protein engineering of various functions including ice binding mimicking insect antifreeze proteins. However, the data presented below demonstrate that the BRD polypeptides undergo ordered and reversible metal ion-dependent protein polymerization in the presence of lanthanum ions. The process generates very long, thin, unbranched filaments that exhibit similarities and differences to classical amyloid fibrils.
La3+ causes ordered aggregation of the BRD polypeptides
The trivalent lanthanide metal lanthanum was substituted for calcium. It has a very similar ionic radius but an additional charge which enables the ion to occupy calcium-binding sites with a higher affinity than calcium . The addition of lanthanum chloride to a solution containing either BRD polypeptide produced a cloudy suspension rendering CD, NMR and fluorescence experiments ineffective.
The addition of lanthanum to other unrelated peptides and proteins did not produce a significant increase in right angle light scattering, suggesting that aggregation of peptides in the presence of lanthanum is not a general phenomenon (Figure 4B). The controls included a 15-residue peptide corresponding to the N-terminal sequence of m-calpain (a cysteine protease) large subunit  (1578.9 Da) (red line in Figure 4B) and calpastatin (CAST), a relatively unstructured 10.5-kDa protein that specifically inhibits calpain  (green line in Figure 4B). The final experiment in Figure 4B shows that a peptide-free control (purple line in Figure 4B) did not produce an increase in right angle light scattering thereby confirming that the observed increases for the BRD polypeptides must be the result of specific peptide-La3+ ion interactions. The addition of EDTA to these control solutions has no effect on the scattering signal. Figure 4C highlights the specificity for lanthanum (blue bar) as other divalent and trivalent metal ions failed to produce large increases in right angle light scattering as a result of protein-metal aggregation.
BRD1 and 2 aggregates are protein filaments
Figure 5C shows an EM grid that was prepared from a solution of BRD2 with calcium ions in place of lanthanum ions and no protein filaments are observed. This confirms that calcium is not capable of causing the BRD polypeptides to aggregate into filaments. Figure 5D is an example of a grid that was prepared from a suspension of BR2-La3+ filaments but was subsequently treated in situ with a solution of EDTA. The filaments that were previously visible have disassociated and are no longer visible by negative staining. This finding supports the right angle light scattering observations of EDTA causing filament dissociation by removing lanthanum ions from the aggregates.
Modelling suggests the BRD filaments are head-to-tail polymers
Metal ions must also play a part in the interaction and there are already four designed metal-binding sites in the turn regions of the monomeric BRD polypeptides. It is possible that extra lanthanum ions bind between two half metal binding sites, composed of the Asp and Asn carbonyl groups not already bound to a metal ions and peptide backbone carbonyls, found in the N and C termini of different monomers, essentially gluing two monomers together in a head-to-tail fashion. There are two Asn side chains and one Glu side chain close to the turn regions in BRD1 and two Asp residues in BRD2 that could form half metal binding sites near the two termini of the monomers. It is harder to imagine metal ions binding between faces of BRD monomers as the only carbonyl donors in this region would be from peptide side chains. The only external carbonyl containing side chains in BRD1 are close to the turns as previously mentioned. There are no external Asp or Glu side chains and only two external Asn side chains on BRD2, and these are both near the first N-terminal turn region.
Our prediction is that the BRD monomers interact via a metal-dependent head-to-tail interaction between the N and C termini of two different monomers. This is the simplest repeating unit that could form very long polymers. In addition, the width of such a repeating unit would be around 3 nm, the width of a single filament measured from EM images. Two models are proposed for the polymerization of BRD2 into a protein filament, as shown in Figure 6B. The first is a head-to-tail repeating unit where the front and back faces of BRD2 are always on the same side which produces a bend in the polymer. The second model is based on the front and back faces of the BRD monomer alternating, which causes a twist. These topologies have been observed by EM. Similar models would also be feasible for BRD1 polymerization.
Testing the filament model with a capping modification
The addition of BioBRD2 to BRD2 severely disrupts filament formation (Figure 7B). Samples all contained a total peptide concentration of 10 μM but were comprised of a mixture of BioBRD2 and BRD2 peptides. Samples contained a 1 to 1 (cyan line), 1.5 to 1 (purple line), 4 to 1 (green line) and 9 to 1 (red line) molar excess of unmodified BRD2. These samples were compared to the BRD2 (blue line) and BioBRD2 (yellow line) data from Figure 7A. Hardly any increase in scattering was observed with the 1 to 1 mixture of BRD2 to BioBRD2 (cyan line) while even at 9 to 1 BRD2 to BioBRD2 (red line) the maximum scattering increase was only 24% of that for the same total concentration of BRD2 alone (blue line). Intermediate ratios of the BRD polypeptides showed a slight increase in scattering in proportion to the amount of BRD2 present but the increase in scattering was unstable as shown by the negative slope for each of the datasets.
Controlling filament length by capping
La3+ ions are present in excess over the BRD polypeptides
Amino acid analysis data for BRD2 protein filaments
A) Amino Acid
B) Picomoles in Sample
C) % of Total
D) Expected % in BRD2 Seq
The BRD filaments are not amyloid fibrils
Finally, the BRD filaments were analyzed for their similarity to amyloid fibrils. Two standard spectrometric assays were used, thioflavin T binding  and congo red binding . BRD2 filaments had much lower fluorescent emission at 482 nm (3.0 × 104 AFU) compared to a sample of Aβ40 amyloid fibrils (1.95 × 105 AFU) in the presence of thioflavin T. In addition, BRD2 filaments did not produce a characteristic spectral shift towards 550 nm upon congo red binding compared to serum amyloid A fibrils (data not shown). The A540nm/A480nm for BRD2 was 0.045 compared to 0.16 for serum amyloid A fibrils. Control experiments using Aβ-40 in the presence and absence of 10 mM LaCl3 demonstrated that lanthanum had no intrinsic affect on amyloid fibril staining using thioflavin T and congo red (data not shown).
It is clear that these BRD polypeptides have some very interesting and unusual properties. In the presence of La3+ they form long, thin, unbranched filaments with some tendency to associate side by side to form bundles up to 40 nm thick and/or become tangled. We propose a model for these based on a simple head-to-tail repeating unit of BRD monomers that interact via the N- and C-terminal regions with lanthanum ions bound to the designed metal-binding sites. Lanthanum ions may also interact with charged side chains between the N and C termini of two different monomers, effectively linking the BRD monomers together. This model allows for the polymerization of the BRD monomer by the formation of very long continuous parallel β-sheets. These long β-sheets can then interact with more lanthanum ions via charged side chains and form bridged interactions with other β-roll filaments, allowing for the formation of filament bundles. Branched filaments would be unlikely to form if this model holds true as the head-to-tail interaction does not allow for two BRD monomers to interact at the C terminus of a single monomer. Experimental data support this suggestion as branched filaments have not been observed by EM.
The addition of EDTA causes the filaments to readily disassociate and dissolve. This is a novel feature as peptide aggregates are often very hard to resolubilize. Amino acid analysis proved that the filaments are proteinaceous, with a profile that matches the composition of the BRD polypeptides. Therefore, the observed increase in right angle light scattering is not simply due to a precipitate of insoluble lanthanum compounds. One explanation for disassembly of the filaments in EDTA would be removal of La3+ from the monomer interface thereby destabilizing the structure of the building block. If this happened randomly along the filament the internal breaks would quickly cause filament disassembly. The exact amount of EDTA required to disassociate the β-roll filaments is unclear but based on right angle light scattering timedrive data in Figure 4, EDTA begins to have an effect relatively early in its introduction to the sample (observed as a decrease in right angle light scattering) but it takes around 240 s for the signal to return to baseline. This is twice the length of the lanthanum injection time course although the lanthanum stock is 1 M whereas the EDTA stock is 0.5 M which suggests an equal amount of EDTA is required. Presumably, EDTA would first bind any free La3+ then begin to remove La3+ ions from the filaments leading to disassociation. The dissolution of some of the BRD filaments under washing conditions suggests that a portion of La3+ is bound weakly, most likely the ions bound between BRD monomers and any non-specifically bound ions. The stability constant (Kstab) of the La3+-EDTA complex is 5.01 × 1015 Mol-1 , and as the dissociation constant (Kd) = 1/Kstab, the Kd for La3+-EDTA is 2.0 × 10-16 Mol-1. This binding is 100,000 times tighter than that between EDTA and Ca2+. EDTA therefore has such a strong affinity for La3+ ions that it will likely outcompete the affinity of La3+ for the designed metal-binding sites in the BRD polypeptides. Even if the lanthanum were bound tightly to the polypeptide, EDTA would be able to form stable complexes and lead to dissociation of the BRD filaments.
The BRD filaments only form in the presence of peptide and lanthanum ions. Several controls where carried out using divalent and trivalent metal ions (Ca2+, Mg2+, Mn2+, and Al3+) and none of these metals gave an increase in right angle light scattering due to filament formation, as shown in Figure 4C. Other lanthanoid metals were tested (Ce3+, Nd3+, Sm3+ and Tb3+) and some of these gave small right angle light scattering increases. However, no filaments were visible by EM (data not shown). It is interesting to note that studies on the R-module of AlgE4 from Azotobactor vinelandii, where the lanthanide thulium was used in place of calcium, gave a precipitate at 6 mM or higher thulium . Thulium ions were also shown to be binding to negative residues on the surface of the R-module and in the unordered C-terminal region. The precipitate was not analyzed further so it is not known if protein filaments formed.
Amino acid analysis and elemental analysis data suggest that around 75 La3+ ions are present for every BRD monomer and not all of the peptide and metal present in solution forms filamentous aggregates. A slightly lower ratio was obtained from right angle light scattering data but lanthanum is clearly in excess and therefore cannot simply be binding just to the four designed calcium-binding sites in the BRD polypeptides. Analysis of stained but unwashed EM grids showed a huge amount of electron density which was easily washed off by rinsing the grids in water before placing them on the EM. This may be excess lanthanum that precipitates with the filaments, artificially raising the amount of lanthanum associated with the peptide filaments.
The BRD filaments are similar in diameter and appearance to some amyloid protofibrils  but do not always form highly ordered bundles. The bundles that do form are not as thick as those seen for amyloid under EM and atomic force microscopy (AFM) [25, 29]. There is also variability in the length of the filaments. Filament length increases as a function of time; longer growth times produce longer fibrils. On a short time scale, the scattering of samples in the presence of lanthanum would slowly increase until the addition of EDTA was initiated, suggesting that the filaments were increasing in size. On a longer time scale, samples that were set up for EM showed a general increase in filament length the longer they were incubated on the EM grids prior to staining. This suggests the filaments are growing from a nucleus and additional BRD monomers are added to the ends of the filament nucleus. In addition, the scattering responses of BRD2 polypeptides were larger than those seen for BRD1 polypeptides at an identical concentration. The BRD2 polypeptide is 16 amino acid longer (~50% larger) than BRD1 and 1.3 kDa larger in mass. The difference in size and mass between these two polypeptides would be magnified in a filament where the units repeat and could explain the higher scattering responses for BRD2 filaments. The models we present in Figure 6 fit the experimental data and the continual addition of further BRD polypeptides via N- to C-terminal interactions could produce filaments that are hundreds of nanometers in length. The model assumes the peptides do adopt a β-roll-like conformation as the diameter of a single filament (~3 nm) closely matches that of the β-roll peptide model (3 nm) in Figure 2.
The modification of BRD2 with an N-terminal biotin residue lends further support to our model of filament formation. The modified peptide can clearly inhibit filament formation and does not form filaments alone. Therefore, the N terminus of the BRD peptide is important in the aggregation process. The ability to exert some control over filament length and abundance by altering the amount of BioBRD2 is of great interest and practical application. This capping strategy could be altered to have other modified BRD polypeptides with biotin or other functional groups positioned elsewhere on the BRD unit which would allow for decoration of the filaments with other moieties such as enzyme subunits, fluorescent tags or antibodies. The degree of decoration could be controlled in an analogous manner to the capping strategy. A higher proportion of modified BRD monomers would lead to more decoration and vice versa. In theory it is possible that a filament could be grown with a core of BRD2 peptides, an N-terminal cap and several different modified BRD polypeptides. Protein and peptide-based nanomaterials are of great interest and have many potential applications, several of which have been reported recently [30–34].
The aggregation of β-sheet rich proteins is not uncommon as β-strands are intrinsically insoluble  and nature has evolved several ways in which to avoid the aggregation of proteins containing β-sheets . The de novo design of soluble β-sheet proteins has proven to be particularly challenging but there have been some notable successes [21, 37–39]. With the challenges of designing soluble β-sheet proteins in mind, it is unsurprising that many designed β-sheet proteins form aggregates (either by misfolding or implicit design) and some of these are fibrillar in nature [40–43]. It is also possible to convert a protein that would normally from ordered aggregates into a soluble monomeric from  or assembly can be blocked by the addition of various inhibitors [45–47]. Our BRD polypeptides are another example of a designed protein that forms an ordered aggregate. However, they can be disassembled simply by the addition of chelating agents such as EDTA and the addition of a biotin residue at the N-terminus can block filament polymerization.
In-register parallel β-sheet arrangements have been demonstrated for several proteins [48–51]. However, these folds do not closely resemble β-rolls as the β-strands of a β-roll are not in-register. Prion protein fibrils have been modelled as β-helical repeating units (similar to β-rolls but the turn regions are less tight) but this model does not stand up to scrutiny as well as the spiral model of prion protein fibrils . Similarly, Sup35 fibrils have also been modelled as β-helices with parallel β-strands similar to those found in β-rolls  but the model has been invalidated . The β-roll model of HET-s fibrils from Podospora anserina  is the most similar model but does not rely on metal-binding and is not reversible. However, there is evidence that metal ions, particularly Zn2+ and Cu2+, are involved in amyloidogenesis and other related pathways in neurodegenerative diseases [55, 56].
In comparison to amyloid fibrils, the BRD filaments are of similar size to amyloid protofilaments, presumably because they share repeating parallel β-strand motifs, but they do not stain with congo red of thioflavin T and they do not associate into fibrils as readily. The BRD filaments are also easily solubilised by the addition of EDTA.
Since beginning this design project it is of interest to note that our lab has discovered a hyperactive antifreeze protein from Antarctic lake bacterium, Marinomonas primoryensis, that appears to be similar to RTX toxin proteins and alkaline protease from Pseudomonas aeruginosa. The protein is very large and contains several domains (unpublished). The domain responsible for the very high antifreeze protein activity contains a putative β-roll with GGXGXDXUX repeats, that is very similar to the alkaline protease β-roll template used to design the filament forming BRD polypeptides. Antifreeze activity is only observed in the presence of Ca2+ suggesting that the domain forms a calcium-bound β-roll motif. The BRD polypeptides were originally designed with the future goal of engineering an antifreeze protein. At the time, no such β-roll AFP had been discovered but our recent findings support the use of a metal-binding β-roll peptide as a scaffold to design an antifreeze protein.
In summary, polypeptides based on the calcium-binding β-roll domain of alkaline protease from Pseudomonas aeruginosa were designed and synthesized. In the presence of lanthanum ions, the polypeptides polymerized and formed non-amyloid-like aggregates. Analysis of this phenomenon by right angle light scattering and electron microscopy confirmed the presence of protein filaments several hundreds of nanometers in length but only 3 nm in diameter. These filaments were only formed in the presence of La3+, no other metal ions tested could induce filament formation. A novel feature of these filaments is their reversible nature caused by the addition of EDTA. In addition, modification of the N terminus of BRD2 with biotin produced a capping effect whereby the length and abundance of the filaments could be controlled by varying the ratio of modified to unmodified peptide.
Peptide synthesis and purification
Peptides were prepared by solid-phase synthesis on a PerSeptive Biosystems 9050 Plus peptide synthesizer, as peptide-amides using PAL-PEG-PS resin (PerSeptive Biosystems). A coupling procedure, employing O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate active esters of 9-fluorenylmethoxy-carbonyl amino acids was used. The peptides were cleaved from the resin with an 81:13:1:5 (v/v) mixture of trifluoroacetic acid, thioanisole, m-cresol, and ethanedithiol, respectively, for 30 min at 25°C. The peptides were purified by C18 reversed-phase chromatography, and peptide identity was confirmed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and amino acid analysis. Peptide purity was assessed by analytical C18 reversed-phase chromatography using the Pharmacia FPLC system. BioBRD2 was synthesized using FMOC solid phase chemistry by Dr. Nam-Chaing Wang at the Advanced Protein Technology Centre, The Hospital for Sick Children, Toronto, ON, Canada. The peptide was purified by HPLC as stated above.
Circular dichroism spectroscopy
Circular Dichroism measurements were performed on an Aviv 62A DS Circular Dichroism Spectrometer. Spectra were obtained using a 1 mm quartz cuvette at 25°C in water or 10 mM HEPES pH 7.6. The peptide concentration for each spectra was 10 μM and the wavelength range was 190–280 nm. All CD measurements are reported in millidegrees.
1D NMR spectroscopy
1D NMR spectra were recorded on a Varian Inova 600 MHz NMR spectrometer equipped with a z-gradient HCN probe. Acquisition parameters were: acquisition time 2 s; sweep width 8000 Hz; recycle delay 2.5 s; number of scans 512; and a 90° pulse. Spectra are presented with a 0.5 Hz line broadening for increased signal-to-noise. The BRD1 sample was added to 90% H2O/10% D2O to make an approximately 0.5 mM solution, and the pH adjusted to 6.5. Calcium was titrated into the sample up to 128 mM.
Right angle light scattering timedrives
Right angle light scattering was measured using a Perkin Elmer LS-50 luminescence spectrometer and FL WinLab v3.0 software. Samples were excited at 320 nm and the right angle light scattering at 320 nm at 90° to the excitation beam was collected. Slit widths were set to 2.5 nm and all experiments were carried out at room temperature (~22°C). Samples were placed in a stirred 3 mL quartz fluorescent cuvette containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.6 as a buffer. At 200 s a solution of BRD polypeptide(s) was injected into the cuvette usually giving a final concentration of 10 mM BRD polypeptide but other concentrations were also used. At 500 s, a 1 M stock of lanthanum trichloride was injected using a syringe pump set to 4 μL/min for a total of 120 s. At 1200 s, a 0.5 M stock solution of EDTA was injected, also at 4 μL/min until the right angle light scattering signal returned to the baseline.
Initial samples of the BRD1 and 2 aggregates were dried for a period of a few days on charged Pioloform, carbon-coated EM grids and then negative stained with 1 % phosphotungstic acid. For subsequent EM images, negatively stained fibrils were prepared by floating peptide solutions (100 μM total peptide concentration, 10 mM La3+ in 20 mM HEPES, pH 7.6) on Formvar-coated Ni2+ EM grids (Pelco No. 160, Redding, CA, USA). These solutions were incubated for 3–5 days. After the grids were blotted and air-dried, the samples were stained with 1 % (w/v) phosphotungstic acid. Electron microscopy images of the peptides were acquired on a Hitachi H-7000 transmission electron microscope (TEM) operated with an accelerating voltage of 75 kV at 30,000 × magnification.
Amino acid analysis and elemental analysis
Amino acid analysis was performed at the Advanced Protein Technology Centre, The Hospital for Sick Children, Toronto, ON, Canada. Trace element analysis for La3+ was carried out on samples using inductively coupled plasma atomic emission Spectra (ICP-AEOS) at ANALEST, Department of Chemistry, University of Toronto, ON, Canada.
BRD polypeptide modeling
The BRD1 and 2 models were constructed and minimized using SYBYL 6.0. The structures of BRD1 and 2 were threaded onto the alkaline protease β-roll domain using Swiss-PdbViewer and SWISS-MODEL. The models of the docked BRD2 filaments were generated using HEX. Ray traced figures in this paper were generated using PyMol v0.99.
Dynamic light scattering
DLS data were collected using a Wyatt DynaPro Titan molecular sizing system at 25°C using Dynamics software v5.26.38. The system was calibrated using ultrapure water and 2 mg/mL bovine serum albumin (BSA). Samples were prepared as above (100 μM final protein concentration) and analyzed in a 12 μL quartz cuvette. A total of 25 data points were collected for calculation of the average hydrodynamic radius for each sample.
Thioflavin T fluorescence scans
Samples of BRD2 filaments (100 μM final peptide concentration, 10 mM La3+) and Aβ-40 amyloid fibrils (with and without 10 mM La3+) were assayed for thioflavin T (ThT) binding (0.035 mg/mL) using a Perkin Elmer LS-50 luminescence spectrometer and FL WinLab v3.0 software. Samples were excited at 455 nm in stirred 1 mL quartz fluorescent cuvettes and spectra were collected between 460 nm and 560 nm (Slit widths = 2.5 nm).
Congo Red UV/Visible spectrometry
Samples of BRD2 (100 μM final peptide concentration) with and without La3+ (10 mM final concentration) and serum amyloid A fibrils purified from rat pancreas were allowed to bind to freshly prepared Congo red (7 μM) overnight in a 1 mL disposable cuvette. The samples were mixed and the spectrum from 400 nm to 600 nm was recorded on a Cary 50 Bio UV spectrophotometer running Cary WinUV v2.0 software.
- BRD1 and 2:
β-roll designed polypeptides 1 and 2
full length calpastatin
dynamic right angle light scattering
nuclear magnetic resonance
This research was principally funded by the Government of Canada's Network of Centers of Excellence program supported by the Canadian Institute for Health Sciences and the Natural Sciences and Engineering Research Council through PENCE (the Protein Engineering Network of Centers of Excellence). Additional funding was from CIHR to PLD, who holds a Canada Research Chair in Protein Engineering. We thank Judy Vanhorne for help with EM studies.
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