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1 Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy
2 Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom
Reprint requests to: Christopher M. Dobson, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK; e-mail: cmd44{at}cam.ac.uk; fax: 44-1223-763418 or Massimo Stefani, Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy; e-mail: stefani{at}scibio.unifi.it; fax: 39-055-4222725.
(RECEIVED March 16, 2001; FINAL REVISION September 18, 2001; ACCEPTED September 19, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.10201.
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Keywords: Aggregation; amyloid fibrils; HypF N-terminal domain; protofilaments; trifluoroethanol
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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Recently, it has been found that proteins other than those associated with diseases are capable of forming amyloid fibrils under appropriate conditions in vitro (Guijarro et al. 1998; Litvinovich et al. 1998; Chiti et al. 1999; Konno et al. 1999; Krebs et al. 2000; Ramirez-Alvarado et al. 2000; Villegas et al. 2000; Yutani et al. 2000; Fandrich et al. 2001; Pertinhez et al. 2001). It has been proposed that amyloid fibril formation can occur when the native globular fold of a protein is destabilized under conditions in which non-covalent interactions still remain favourable (Chiti et al. 1999). The ability for rational design of conditions promoting amyloid formation has implications for understanding the origin of amyloid formation in vivo because the latter can no longer be assumed to arise from the unique physical properties of a limited number of protein sequences (Chiti et al. 1999; Dobson 1999; Rochet and Lansbury 2000). In addition, the possibility of producing amyloid fibrils under controlled conditions provides us with the opportunity to investigate the molecular basis of amyloid formation using a wide range of proteins.
In this work, we investigate the conditions favorable for amyloid fibril formation using the N-terminal domain of HypF, a newly cloned globular protein factor participating in the maturation of the prokaryotic [NiFe] hydrogenase that is involved in hydrogen metabolism (Friedrich and Schwartz 1993). In addition to showing that this domain is capable of forming in vitro amyloid fibrils of the type associated with diseases, we will describe how solution conditions can be designed to promote selective formation of either the mature fibrils typical of pathological states or their constituents, the protofilaments that normally associate laterally or twist together to form the mature fibrils.
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shows the far-UV circular dichroism (CD) spectra of the purified HypF N-terminal domain acquired in the presence and absence of 5 M GdnHCl (guanidine hydrochloride). Unlike the CD spectrum acquired in the presence of GdnHCl, the spectrum of the protein acquired under native conditions is consistent with a well-structured protein with an 
/ß topology. Figures 1B,C show the change of mean residue ellipticity at 222 nm on the increase of urea concentration and temperature, respectively. The urea and heat-denaturation curves reveal single sharp transitions, indicating that this protein fragment has a globular and well-defined three-dimensional structure under native conditions. The analysis of the urea denaturation curve yielded values of 29±3 kJ/mol, 7±1 kJ/mol per mole, and 4.2±0.2 for the 
G(H2O), m, and Cm values, respectively. Unlike the urea-denatured protein, the heat-denatured domain was not capable of recovering native-like ellipticity when cooled to room temperature, indicating that the thermal denaturation process is irreversible. Therefore, determination of the thermodynamic parameters characterising the unfolding thermal reaction was not attempted.
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). Analysis of 
100 fibrils from three different specimens identified three different types of fibrils, each displaying a characteristic width: 3–5 nm, 7–9 nm, and 12–20 nm. Figure 2B shows an area of a specimen in which the three different types are all present (numbered 1, 2, and 3, respectively). An 8 nm fibril shown in the figure (numbered 2) appears to dissociate at its terminus into its constituents, allowing a 4 nm fibril to be observed (numbered 1). Fig. 2C shows another detail in which two fibrils of ca. 8 nm appear to associate and twist together to form a larger fibril. In other cases, pairs of 8 nm fibrils seem to associate in a parallel manner forming 15–20 nm ribbon-like fibrils (Fig. 2A–B). Although the finer structure of the three types of fibrils is not visible from these micrographs, these pictures indicate that the fibrils with 12–20 nm widths originate from the association of pairs of fibrils of 7–9 nm in width which, in turn, result from the assembly of two or more fibrils 3–5 nm in width. The latter are extremely rare in the samples incubated at low pH values and are visible only at the ends of some of the fibrils with diameters of 7–9 nm when these appear to dissociate into their constituents. Many studies have regarded fibrils of 2–5 nm in width as protofilaments, (i.e., the basic units that assemble to form larger fibrils; Glenner et al. 1971; Merz et al. 1983; Serpell et al. 1995; Goldsbury et al. 1997; Harper et al. 1997; Conway et al. 1998; Serpell et al. 2000). Hence, it is likely that the narrowest fibrils observed here represent the protofilaments that associate to form the larger fibrils. However, one cannot rule out that the 3–5 nm fibrils also originate from the association of narrower filaments.
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), but with a different size distribution. The 3–5 nm and 7–9 nm wide fibrils are both abundant, whereas the largest ones (12–20 nm) appear to be completely absent altogether. Figures 2D,E show regions of specimens revealing many examples of fibrils with diameters of 3–5 and 7–9 nm, respectively. Figure 2F shows another region in which both types of fibrils are present (numbered 1 and 2). Unlike the situation in the samples incubated at low pH, the narrowest fibrils grown in the presence of TFE are abundant and appear fully dissociated from the fibrils with larger widths. Some of these are short and curved, resembling the fibrillar aggregates called protofibrils observed for other protein systems (Harper et al. 1997). In contrast, others are long and rigid resembling more closely the fibril constituents (i.e., protofilaments). Interestingly, a size distribution of this type was also obtained when TFE was added to fibrils grown at pH 3.
The aggregates of the HypF N-terminal domain formed in the presence of TFE were analysed further with two colorimetric assays specific for amyloid fibrils. Samples incubated for one month in the presence of moderate concentrations of TFE cause the enhancement of fluorescence of Thioflavine T (ThT). This enhancement is more than 30 times higher than that observed on addition of a protein sample incubated in the absence of TFE (Fig. 3). The absorption peak of Congo red also undergoes a red-shift when aliquots of the protein solutions incubated under these conditions are added to the dye (Fig. 4). An increase of the absorbance values throughout the whole range of wavelength investigated here is also observed when the protein aggregates are added to the dye. This increase clearly originates from the light scattering by the protein aggregates; indeed, the spectrum of the aggregated protein in the absence of Congo red yields absorbance values higher than zero (Fig. 4, dotted line). Subtraction from the experimentally measured spectrum of the aggregates in the presence of Congo red of spectra of the aggregates alone and Congo red alone yields a difference spectrum with a maximum at 530–540 nm (Fig. 4, inset). Such a result is expected only if ordered amyloid-like aggregates are present in the solution (Klunk et al. 1989). Figure 5 shows the time-course of the change of ThT fluorescence as the protein aggregates form at pH 3 or in the presence of TFE. Whereas at acidic pH the aggregates develop after a lag phase of several days, in the presence of TFE the maximum ThT fluorescence is reached after a few minutes (Fig. 5, inset). The observed kinetics indicate that the prevalence of fibrils with larger diameters in the samples incubated at low pH cannot be attributed to a more rapid rate of the fibril formation process.
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Discussion |
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Furthermore, using the HypF N-terminal domain we have shown that it is possible to select solution conditions to promote formation of either amyloid fibrils or their constituent protofilaments. Moderate concentrations of TFE destabilize the interactions promoting the assembly of protofilaments formed from this protein domain. It was reported previously that long-term incubation of acylphosphatase in the presence of moderate concentrations of TFE also generates relatively long protofilaments that remain dissociated for periods longer than those observed for other fibril formation processes (Chiti et al. 1999). Furthermore, addition of TFE to fibrils obtained from the peptide corresponding to the 10–19 region of the transthyretin sequence disrupts the rigid fibrils to reveal the constituent protofilaments (MacPhee and Dobson 2000). A peptide corresponding to the 34–42 region of the sequence of the Aß fragment associated with Alzheimer's disease generates protofilaments, rather than complete fibrils, when the hydrophobic residues that are thought to be exposed on the surface of the protofilament are replaced with hydrophilic ones (Lazo and Downing 1999). Finally, protofilaments are also more stable when the L55P mutant of transthyretin undergoes aggregation (Lashuel et al. 1999).
All these findings, in addition to the results reported here with the HypF N-terminal domain, indicate that protofilament association into larger fibrils is likely to be guided, at least in part, by interactions between the side chains of hydrophobic residues that remain solvent-exposed on the surface of individual protofilaments and that mild destabilization of such interactions makes it possible to form and isolate the individual protofilaments. The ability of TFE to stabilize mainchain hydrogen bonds within individual protofilaments may also contribute to the relative stability of these structures in solutions containing TFE, as ß-sheet structure is probably an important force driving the assembly of individual protein or peptide molecules into protofilaments. Protofilaments can grow and be stable independently of any further supramolecular organization. Solutions containing moderate concentrations of TFE are very favorable for the stabilization of individual protofilaments as TFE is known to weaken hydrophobic interactions without disrupting the backbone hydrogen bonds within individual protofilaments. Although we cannot make the generalization that TFE leads universally to protofilament formation (as opposed to other partially denaturing conditions such as low pH, high temperatures, and destabilizing mutations), we propose that dissociation of fibrils into their constituent protofilaments requires conditions destabilizing hydrophobic interactions that might exist at the interface between interacting protofilaments.
The ability for rational design of suitable solvent conditions for the formation of individual protofilaments has implications for the investigation of the structure and the mechanism of formation of amyloid fibrils. The possibility of preparing isolated protofilaments to order will allow structural investigations to be carried out on materials with a lower degree of complexity than mature fibrils. It will also facilitate the biophysical investigation of the steps associated with aggregation as protofilament formation is likely to be a less complex process than full fibril formation. More importantly, experimental procedures directed to the formation of protein aggregates and fibrils of various types will help in the identification of the aggregate species responsible for cell damage.
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Materials and methods |
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cells by Genomic PrepTM (Pharmacia). The DNA fragment corresponding to residues 993–1263 of the whole hypf gene was amplified by PCR using suitable primers containing the restriction sites for BamHI and EcoRI, respectively. Samples (1.0 µM) of each primer were added to 100 ng of template DNA, 10X PCR buffer, 200 µM dNTPs, and 2.5 units of Taq DNA polymerase (Finnzymes) in a volume of 50 µL. The fragments resulting from PCR amplification (94°C for 15 s, 52°C for 1 min, 72°C for 1 min, 25 cycles) were digested with BamHI and EcoRI, ligated into pGEX-2T downstream and in frame with glutathione S-transferase, and sequenced in their entirety. Expression of protein in the E. coli DH5 cells and its subsequent purification were carried out as described previously for muscle acylphosphatase (Modesti et al. 1995). Protein purity and quality were checked by SDS-PAGE, ES-MS, and amino acid analysis. The resulting sequence of the domain is AKNTSCGVQLRIRGKVQGVGFR PFVWQLAQQLNLHGDVCNDGDGVEVRLREDPETFLVQLY QHCPPLARIDSVEREPFIWSQLPTEFTIR. A Gly-Ser dipeptide is also present at the N terminus as a result of the cloning in pGEX-2T.
Circular dichroism
Far-UV CD spectra were acquired using a Jasco J-720 spectropolarimeter with a thermostat (Great Dunmow, Essex, UK). The CD spectra were recorded at a protein concentration of 0.2 mg mL-1 in 50 mM acetate buffer (pH 5.5), 25°C. To acquire the CD spectrum of the domain under denaturing conditions, guanidinium chloride was added to a final concentration of 5.5 M.
Equilibrium urea- and heat-denaturation
25 samples containing 0.2 mg/mL protein in 50 mM acetate buffer (pH 5.5) were equilibrated at 25°C in the presence of different concentrations of urea ranging from 0 to 8 M. The mean residue ellipticity at 222 nm was plotted versus urea concentration and the resulting curve analyzed using the method described by Santoro and Bolen (1988). For acquiring a heat-denaturation curve, a 0.2 mg/mL solution of the N-terminal HypF domain, incubated in acetate buffer (pH 5.5), was placed in the CD cell holder and the temperature increased slowly at a rate of 0.5°C/min. A thermocouple was used to monitor the temperature inside the cuvette and the CD signal at 222 nm was acquired at 1.0°C intervals from 20° to 76°C.
Electron microscopy
Electron micrographs were acquired using a JEM 1010 transmission electron microscope at 80 kV excitation voltage. In each case, a 3 µL sample of protein solution was placed on a formvar- and carbon-coated grid. The sample was then negatively stained with 30 µL of 2% uranyl acetate and observed at a magnification of 12–30,000x. Morphological investigation and size determination of the fibrils were performed by analysing 30–40 fibrils per specimen and 3 specimens for each experimental condition (to achieve a total number of 100 fibrils for each experimental condition).
Thioflavine T assay
The protein domain was incubated for either one week or one month at a concentration of 0.8 mg/mL in 50 mM acetate buffer, 30% (v/v) TFE (pH 5.5) at room temperature. An aliquot of this sample and an aliquot of a highly concentrated solution of ThT were added to a solution of 25 mM phosphate buffer (pH 6.0). Final conditions after mixing were 0.03 mg/mL protein, 25 µM ThT, and 25 mM phosphate buffer (pH 6.0) at 25°C. The fluorescence spectra were acquired immediately after mixing using a Shimadzu RF-5000 spectrofluorimeter and excitation and emission wavelengths of 440 and 485 nm, respectively. For the kinetic experiments, the protein domain was incubated at 0.4 mg/mL at room temperature in 30% (v/v) TFE, 50 mM acetate buffer (pH 5.5) or in 20 mM citric acid (pH 3). At regular time intervals, aliquots of the protein samples were withdrawn to perform the ThT assay as described above.
Congo red assay
The protein was incubated for either one week or one month at a concentration of 0.1 mg/mL in 50 mM acetate buffer, 30% (v/v) TFE (pH 5.5). 133 µL of this sample were mixed with 867 µL of a solution containing 20 µM Congo red, 10 mM phosphate buffer, 150 mM NaCl (pH 7.4). Mixtures without protein and mixtures without Congo red were also prepared as controls. The absorption spectra were acquired after 2–3 min equilibration using an Ultrospec 2000 spectrophotometer (Pharmacia Biotech).
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Acknowledgments |
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References |
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