Aggregation of the Acylphosphatase from Sulfolobus solfataricus
2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês
10.1074/jbc.m312961200
ISSN1083-351X
AutoresGeorgia Plakoutsi, Niccolò Taddei, Massimo Stefani, Fabrizio Chiti,
Tópico(s)Enzyme Structure and Function
ResumoProtein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures. Protein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures. Protein aggregation is a hallmark of a number of human pathologies including Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases and the systemic amyloidoses associated with immunoglobulin light chain, transthyretin, lysozyme, and β2-microglobulin (1Stefani M. Dobson C.M. J. Mol. Med. 2003; 81: 678-699Google Scholar). Under these pathological conditions, a protein or peptide that is normally soluble deposits into fibrillar aggregates commonly referred to as amyloid fibrils; these display a diameter of 7-13 nm, an extensive β-sheet structure, and characteristic staining properties such as a Congo red birefringence under cross-polarized light (2Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Google Scholar). It has long been recognized that the conformational state of a polypeptide chain that forms the precursor aggregates, eventually leading to amyloid formation, is neither the natively folded nor the fully unfolded state but a partially folded state that contains significant structure, although lacking a well defined tertiary fold (3Kelly J.W. Curr. Opin. Struct. Biol. 1998; 8: 101-106Google Scholar, 4Rochet J.C. Lansbury Jr., P.T. Curr. Opin. Struct. Biol. 2000; 10: 60-68Google Scholar). For the majority of the proteins that have been studied in detail, it is clear that this is the case. Fibril formation from transthyretin, for example, occurs preferentially at mildly acidic pH values (5Lai Z. Colon W. Kelly J.W. Biochemistry. 1996; 35: 6470-6482Google Scholar). Under these conditions the native tetrameric conformation of the protein is found to dissociate and to partially unfold, thus adopting a conformation that is highly prone to aggregate and to form amyloid fibrils (5Lai Z. Colon W. Kelly J.W. Biochemistry. 1996; 35: 6470-6482Google Scholar). Similarly, β2-microglobulin forms amyloid fibrils under conditions in which the N- and C-terminal β-strands of the protein are unfolded (6McParland V.J. Kalverda A.P. Homans S.W. Radford S.E. Nat. Struct. Biol. 2002; 9: 326-331Google Scholar). Observations that these regions of the sequence are solvent-exposed in the fibrils formed from this protein provide direct evidence that fibril formation does not originate from the assembly of protein molecules in their native conformation (7Hoshino M. Katou H. Hagihara Y. Hasegawa K. Naiki H. Goto Y. Nat. Struct. Biol. 2002; 9: 332-336Google Scholar). Some other proteins such as cystatin C, which is associated with a hereditary form of cerebral amyloid angiopathy, and the cell cycle regulatory protein p13suc1 are suggested to fibrillize via domain swapping (8Rousseau F. Schymkowitz J.W. Wilkinson H.R. Itzhaki L.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5596-5601Google Scholar, 9Staniforth R.A. Giannini S. Higgins L.D. Conroy M.J. Hounslow A.M. Jerala R. Craven C.J. Waltho J.P. EMBO J. 2001; 20: 4774-4781Google Scholar). Domain swapping is a mechanism in which part of the structure of each monomer replaces the corresponding structural elements of an identical monomer so as to form an oligomer where each subunit has a similar structure to the folded monomer (8Rousseau F. Schymkowitz J.W. Wilkinson H.R. Itzhaki L.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5596-5601Google Scholar, 9Staniforth R.A. Giannini S. Higgins L.D. Conroy M.J. Hounslow A.M. Jerala R. Craven C.J. Waltho J.P. EMBO J. 2001; 20: 4774-4781Google Scholar). Even in this case, aggregation is proposed to require a substantial unfolding of the folded monomer in order to facilitate exchange of structural elements (8Rousseau F. Schymkowitz J.W. Wilkinson H.R. Itzhaki L.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5596-5601Google Scholar, 9Staniforth R.A. Giannini S. Higgins L.D. Conroy M.J. Hounslow A.M. Jerala R. Craven C.J. Waltho J.P. EMBO J. 2001; 20: 4774-4781Google Scholar). The presence of structure in the fibrils formed from the amyloid β peptide associated with Alzheimer's disease indicates that fibrils formed from peptides or proteins that are unstructured in their soluble form need to adopt a partially structured conformation either before or during assembly (10Petkova A.T. Ishii Y. Balbach J.J. Antzutkin O.N. Leapman R.D. Delaglio F. Tycko R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16742-16747Google Scholar, 11Torok M. Milton S. Kayed R. Wu P. McIntire T. Glabe C.G. Langen R. J. Biol. Chem. 2002; 277: 40810-40815Google Scholar). These are just a few examples that support the “conformational change hypothesis” of fibril formation. Nevertheless, the general validity of these theories has been challenged by a number of recent findings. On the one hand, amyloid formation may occur readily from peptides as short as 5 residues, where formation of structure before assembly is rather unlikely given the short length of the peptides (12Lopez de La Paz M. Goldie K. Zurdo J. Lacroix E. Dobson C.M. Hoenger A. Serrano L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16052-16057Google Scholar). On the other hand, a number of observations have indicated that fibrillar aggregates may originate from the assembly of globular protein molecules in their native or native-like state (13Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 17: 2903-2911Google Scholar, 14Laurine E. Gregoire C. Fandrich M. Engemann S. Marchal S. Thion L. Mohr M. Monsarrat B. Michel B. Dobson C.M. Wanker E. Erard M. Verdier J.M. J. Biol. Chem. 2003; 278: 51770-51778Google Scholar). The yeast prion Ure2p was shown to form fibrils that display birefringence upon Congo red binding and increased resistance to proteinase K treatment, typical of amyloid fibrils (15Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1343Google Scholar, 16Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar, 17Thual C. Bousset L. Komar A.A. Walter S. Buchner J. Cullin C. Melki R. Biochemistry. 2001; 40: 1764-1773Google Scholar). It was shown that Ure2p retains its native α-helical content and ability to bind glutathione after assembly into fibrils (13Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 17: 2903-2911Google Scholar). Accordingly, x-ray fiber diffraction analysis has shown that the fibrils do not possess a cross-β-structure (18Bousset L. Briki F. Doucet J. Melki R. J. Struct. Biol. 2003; 141: 132-142Google Scholar). Similarly, lithostathine, a protein that forms both independent and colocalized deposits with amyloid β plaques, neurofibrillary tangles, and PrPsc plaques, maintains its native content of secondary structure, although it aggregates into fibrillar structures (14Laurine E. Gregoire C. Fandrich M. Engemann S. Marchal S. Thion L. Mohr M. Monsarrat B. Michel B. Dobson C.M. Wanker E. Erard M. Verdier J.M. J. Biol. Chem. 2003; 278: 51770-51778Google Scholar). In this case the resulting proteinase K-resistant fibrils do not bind Congo red in addition to showing no cross-β-structure (14Laurine E. Gregoire C. Fandrich M. Engemann S. Marchal S. Thion L. Mohr M. Monsarrat B. Michel B. Dobson C.M. Wanker E. Erard M. Verdier J.M. J. Biol. Chem. 2003; 278: 51770-51778Google Scholar). The ability of these two proteins to form fibrillar structures under non-denaturing conditions close to a physiological medium suggests that a substantial unfolding is not required (13Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 17: 2903-2911Google Scholar, 14Laurine E. Gregoire C. Fandrich M. Engemann S. Marchal S. Thion L. Mohr M. Monsarrat B. Michel B. Dobson C.M. Wanker E. Erard M. Verdier J.M. J. Biol. Chem. 2003; 278: 51770-51778Google Scholar). Amyloid formation is a property that is not only shared by a few polypeptide chains associated with disease. Following the initial observation that the Src homology 3 domain from phosphatidylinositol 3-kinase is able to form fibrillar aggregates structurally indistinguishable from those formed naturally under pathological conditions (19Guijarro J.I. Sunde M. Jones J.A. Campbell I.D. Dobson C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4224-4228Google Scholar), ∼20 proteins were shown to have the potential to form amyloid fibrils under appropriate conditions in vitro (1Stefani M. Dobson C.M. J. Mol. Med. 2003; 81: 678-699Google Scholar). In addition to a rise in general awareness that protein sequences, and more generally the biology of cells, have evolved structural adaptations and biological mechanisms to effectively defend from undesired protein aggregation, these findings have provided new model systems to investigate the fundamentals of protein aggregation (1Stefani M. Dobson C.M. J. Mol. Med. 2003; 81: 678-699Google Scholar). Amyloid formation from these non-pathological systems occurs under mild denaturing conditions, such as at low pH values, at high temperatures and in the presence of organic cosolvents. Moreover, mutations that destabilize the native conformation of a protein increase its propensity to aggregate (20Chiti F. Taddei N. Bucciantini M. White P. Ramponi G. Dobson C.M. EMBO J. 2000; 19: 1441-1449Google Scholar, 21Ramirez-Alvarado M. Merkel J.S. Regan L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8979-8984Google Scholar). These observations support further the view that substantial unfolding of the compact globular state of a protein is generally the first conformational change that initiates aggregation. In this study, we show that aggregation of one of these non-pathological proteins may result from a conformational state that presents only minor structural modifications compared with the native form of the protein. The protein that we have used is the acylphosphatase from the hyperthermophile Archaea Sulfolobus solfataricus (Sso AcP). 1The abbreviations used are: Sso AcP, S. solfataricus acylphosphatase; ANS, 1-anilino-8-naphthalenesulfonic acid; DLS, dynamic light scattering; GdnHCl, guanidine hydrochloride; TFE, 2,2,2-trifluoroethanol; ThT, thioflavine T. 1The abbreviations used are: Sso AcP, S. solfataricus acylphosphatase; ANS, 1-anilino-8-naphthalenesulfonic acid; DLS, dynamic light scattering; GdnHCl, guanidine hydrochloride; TFE, 2,2,2-trifluoroethanol; ThT, thioflavine T. A stable protein from a hyperthermophilic organism is likely to maintain its native topology under a wide range of conditions, including relatively harsh solvent conditions that normally promote aggregation. Utilization of a hyperthermophilic protein therefore allows aggregation processes to be investigated under conditions in which the native conformation is not significantly disrupted. Although the native structure of Sso AcP has not yet been resolved, the relatively high sequence identity with the previously characterized human muscle and common-type acylphosphatases and the N-terminal domain of the Escherichia coli HypF suggests it may display the same basic ferredoxin-like topology, consisting of a five-stranded antiparallel β-sheet facing two antiparallel α-helices (22Pastore A. Saudek V. Ramponi G. Williams R.J. J. Mol. Biol. 1992; 224: 427-440Google Scholar, 23Thunnissen M.M. Taddei N. Liguri G. Ramponi G. Nordlund P. Structure. 1997; 5: 69-79Google Scholar, 24Rosano C. Zuccotti S. Bucciantini M. Stefani M. Ramponi G. Bolognesi M. J. Mol. Biol. 2002; 321: 785-796Google Scholar). In addition to illustrating the capability of this protein to form ordered aggregates, we will present kinetic evidence that aggregation originates from a conformational state that retains enzymatic activity and is located on the native side of the major free energy barrier of unfolding. Materials—Thioflavine T (ThT), Congo red, 1-anilino-8-naphthalenesulfonic acid (ANS), guanidine hydrochloride (GdnHCl), and 2,2,2-trifluoroethanol (TFE) were purchased from Sigma. Benzoyl phosphate was synthesized and purified as described (25Camici G. Manao G. Cappugi G. Ramponi G. Experientia (Basel). 1976; 32: 535-536Google Scholar). Cloning, Expression, and Purification of Sso AcP—The DNA fragment corresponding to the Sso AcP gene, originally inserted in a pEMBL plasmid, was amplified by PCR using two primers that contained the restriction sites for BamHI and EcoRI. The resulting amplified fragments were purified using the QiaQuick Purification Kit by Qiagen (Milano, Italy), digested with BamHI and EcoRI for 2 h, purified again, and then ligated into pGEX-2T plasmid, previously digested with the same restriction enzymes. The resulting plasmid was checked by DNA sequencing and then transformed into E. coli DH5α cells. Gene expression in the DH5α cells and purification of the resulting protein were carried out as described for human muscle acylphosphatase (26Taddei N. Stefani M. Magherini F. Chiti F. Modesti A. Raugei G. Ramponi G. Biochemistry. 1996; 35: 7077-7083Google Scholar). Protein purity was checked by SDS-PAGE and electrospray mass spectrometry. The resulting protein sequence is GSMKKWSDTEVFEMLKRMYARVYGLVQGVGFRKFVQIHAIRLGIKGYAKNLPDGSVEVVAEGYEEALSKLLERIKQGPPAAEVEKVDYSFSEYKGEFEDFETY. The Gly-Ser dipeptide at the N terminus results from the cloning in pGEX-2T. The underlined Met residue is residue 1. Purified protein was stored in 10 mm Tris, pH 8.0. Protein concentration was determined by UV absorption using an ϵ280 value of 1.26 ml mg-1 cm-1. Thioflavine T Assay—Sso AcP was incubated at a concentration of 0.4 mg ml-1 for 2 h in 50 mm acetate buffer, pH 5.5, 25 °C in the presence of various concentrations of TFE. 60 μl of each sample were added to 440 μl of a solution containing 25 μm ThT, 25 mm phosphate buffer, pH 6.0. The fluorescence of the resulting samples was measured at 25 °C using a 2 × 10-mm path length cuvette and a PerkinElmer LS 55 spectrofluorimeter equipped with a thermostated cell compartment. The excitation and emission wavelengths were 440 and 485 nm, respectively. The kinetics of aggregation of Sso AcP were studied by incubating the protein under the same conditions of pH and temperature in 15, 20, 25, 50, and 70% of TFE (v/v). Aliquots were withdrawn at regular time intervals and used for the ThT assay as described above. In order to determine the rate constants of aggregation (k), the plots of the fluorescence intensity versus time were fit to single exponential functions of the form y = q + A exp(-kt). Congo Red Assay—Sso AcP was incubated at a concentration of 0.4 mg ml-1 for 30 min in 50 mm acetate buffer, pH 5.5, 25 °C in the presence of 0, 20, 50, or 70% (v/v) TFE. Aliquots of 60 μl of each protein solution were mixed with 440 μl of solutions containing 20 μm Congo red, 5 mm phosphate buffer, 150 mm NaCl, pH 7.4. After a 2-3-min equilibration, optical absorption spectra were acquired from 400 to 700 nm by an Ultrospec 2000 UV-visible spectrophotometer (Amersham Biosciences). A 4 × 5-mm path length cuvette was used. Solutions without protein and solutions without Congo red were used as controls. ANS Assay—Sso AcP was incubated for 30 min at 25 °C as described above for the ThT assay. Aliquots of 60 μl of the protein solutions were mixed with 440 μl of solutions containing 1 mm ANS, in 50 mm acetate buffer, pH 5.5, 25 °C. Fluorescence spectra were acquired using the PerkinElmer LS 55 spectrofluorimeter, an excitation wavelength of 390 nm, and an emission range from 410 to 620 nm. A 2 × 10-mm path length cell was used. Dynamic Light Scattering—Sso AcP was incubated for 30 min as described above for the ThT assay. Size distributions of the protein samples by intensity were obtained before and after centrifugation of the samples (18,000 rpm for 10 min). The data were attained using a Zetasizer Nano S dynamic light scattering (DLS) device from Malvern Instruments (Malvern, Worcestershire, UK). The temperature was maintained at 25 °C by a thermostating system. Low volume 12.5 × 45-mm disposable cells were used. The buffer was filtered immediately before use to eliminate any impurities. Far-UV Circular Dichroism—Sso AcP was incubated for 30 min as described above for the ThT assay. Far-UV CD spectra were acquired at 25 °C using a cuvette of 1-mm path length and a Jasco J-810 spectropolarimeter (Great Dunmow, Essex, UK) equipped with a thermostated cell holder. Each spectrum was recorded as the average of three scans. The first set of spectra for the samples was acquired after an incubation of 30 min at 25 °C. A second set of spectra was acquired after centrifugation of the samples at 18,000 rpm for 10 min. In the kinetic experiment the far-UV ellipticity at 222 nm was monitored for Sso AcP at 0.4 mg ml-1 in 20% (v/v) TFE at 25 °C for 1 h. Electron Microscopy—Electron micrographs were acquired by a Joel JEM 1010 transmission electron microscope (Tokyo, Japan) at 80-kV excitation voltage. The samples consisted of 0.4 mg ml-1 Sso AcP incubated for 1 h at 25 °C in 50 mm acetate buffer, pH 5.5, in the absence or presence of 20% (v/v) TFE. 3 μl of the sample were placed on a Formvar and carbon-coated grid from Agar Scientific (Stansted, Essex, UK) for 3-5 min and then blotted off. 3 μl of 2% uranyl acetate were then added, remained on the grid for 3 min, and blotted off. The magnification was 20,000-30,000 times. Enzymatic Activity Measurements—Sso AcP was incubated at a concentration of 0.03 mg ml-1 for 30 min in 50 mm acetate buffer, pH 5.5, 25 °C. Incubation was carried out as follows: (i) in the presence of various concentrations of TFE, (ii) in the absence of TFE, and (iii) in the presence of 6 m GdnHCl. After pre-incubation, enzymatic activity was measured spectrophotometrically using benzoyl phosphate as a substrate (25Camici G. Manao G. Cappugi G. Ramponi G. Experientia (Basel). 1976; 32: 535-536Google Scholar). Briefly, 50 μl of the protein sample were mixed with 950 μl of a solution containing 2.5 mm benzoyl phosphate, 50 mm acetate buffer, pH 5.5, and various TFE concentrations. Final protein concentration was 0.0015 mg ml-1. For the first set of experiments (i) the protein sample was pre-incubated in the same TFE concentration that was used afterward for the enzymatic activity assay. All recorded enzymatic activity values were subtracted by the spontaneous, non-catalyzed hydrolysis of benzoyl phosphate measured under the same conditions. Stopped-flow Kinetics—Folding and unfolding of Sso AcP were followed with a Bio-logic SFM-3 stopped-flow device coupled to a fluorescence detection system (Claix, France) and thermostated with a water-circulating bath. The excitation wavelength was 280 nm, and the emitted fluorescence above 320 nm was monitored using a bandpass filter. For the unfolding experiments, 1 volume of 0.4 mg ml-1 Sso AcP in buffer was mixed with 19 volumes of a solution containing TFE. Folding was initiated by mixing 1 volume of 0.4 mg ml-1 Sso AcP denatured in 6 m GdnHCl with 19 volumes of a solution without GdnHCl and containing low concentrations of TFE. The final conditions were 0.02 mg ml-1 Sso AcP, 50 mm acetate buffer, pH 5.5, 25 °C, and the final TFE concentration range was from 45 to 60% (v/v) (for unfolding) or from 0 to 18% (v/v) (for folding). The dead time was generally 10.4 ms. The kinetic traces were fitted to simple exponential functions of the form shown in Equation 1, y(t)=at+b+Aexp(-kt)(Eq. 1) where y(t) is the fluorescence signal recorded as a function of time; A and k are the amplitude and rate constant, respectively. The additional straight line a t + b was considered to account for trends of the fluorescence signal arising due to aggregation, proline isomerization, fluorescence decay of the sample, and other disturbing factors. Aggregation of Sso AcP was promoted by adding TFE. In addition to be a cosolvent commonly used to promote amyloid formation (20Chiti F. Taddei N. Bucciantini M. White P. Ramponi G. Dobson C.M. EMBO J. 2000; 19: 1441-1449Google Scholar, 27Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Massimo S. Nature. 2002; 416: 507-511Google Scholar, 28Srisailam S. Kumar T.K. Rajalingam D. Kathir K.M. Sheu H.S. Jan F.J. Chao P.C. Yu C. J. Biol. Chem. 2003; 278: 17701-17709Google Scholar), TFE has also proven useful for revealing details of the mechanism of protein aggregation (29Barrow C.J. Zagorski M.G. Science. 1991; 253: 179-182Google Scholar, 30Fezoui Y. Teplow D.B. J. Biol. Chem. 2002; 277: 36948-36954Google Scholar, 31Chiti F. Stefani M. Taddei N. Ramponi G. Dobson C.M. Nature. 2003; 424: 805-808Google Scholar). For the study of aggregation of Sso AcP, we focused on the first aggregational events, i.e. the conversion of the soluble form into pre-fibrillar aggregates. Indeed, several studies suggest that these pre-fibrillar structures may be the key oligomeric species that are toxic to the cells and responsible for the onset of neurodegenerative diseases (27Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Massimo S. Nature. 2002; 416: 507-511Google Scholar, 32Goldberg M.S. Lansbury P.T Nat. Cell Biol. 2000; 2: E115-E119Google Scholar, 33Sousa M.M. Cardoso I. Fernandes R. Guimaraes A. Saraiva M.J. Am. J. Pathol. 2001; 159: 1993-2000Google Scholar, 34Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Google Scholar). Aggregate Formation Monitored by Dye Binding—Sso AcP was incubated for 2 h at 25 °C, pH 5.5, in various TFE concentrations. The samples were then subjected to the ThT assay so as to reveal the presence of aggregates with a relatively ordered organization (35LeVine III, H. Methods Enzymol. 1999; 309: 274-284Google Scholar). Although the samples incubated in the absence or presence of low TFE concentrations did not show an increase in ThT fluorescence, samples incubated at TFE concentrations in the range of 15-35% (v/v) produced a 6-fold increase (Fig. 1). TFE concentrations higher than 40% (v/v) led to a decrease in fluorescence intensity, yet not reaching values of zero. At the highest TFE concentration used (70% (v/v)), the intensity was about half of the maximum value. ANS is a hydrophobic dye that binds to solvent-exposed hydrophobic surfaces of a polypeptide chain (36Semisotnov G.V. Rodionova N.A. Razgulyaev O.I. Uversky V.N. Gripaoe A.F. Gilmanshin R.I. Biopolymers. 1991; 31: 119-128Google Scholar). Whereas folded or unfolded states of proteins exhibit very weak binding affinity, conformational states with clusters of exposed hydrophobic groups feature a much higher affinity allowing this dye to serve as a marker for partially folded states (36Semisotnov G.V. Rodionova N.A. Razgulyaev O.I. Uversky V.N. Gripaoe A.F. Gilmanshin R.I. Biopolymers. 1991; 31: 119-128Google Scholar). Recent reports (37Kremer J.J. Pallitto M.M. Sklansky D.J. Murphy R.M. Biochemistry. 2000; 39: 10309-10318Google Scholar) have shown that aggregates of the amyloid β peptide able to interact with the lipid bilayer of cell membranes are also able to bind and increase the fluorescence of ANS, most probably as a result of the exposure of hydrophobic clusters on the surface of these aggregated structures. Sso AcP was incubated for 30 min under the same conditions used for the ThT measurements. The samples were then used for the ANS assay. At TFE concentrations in the 15-25% (v/v) range, the ANS fluorescence emission was remarkably more intense than that of samples containing no protein (Fig. 2). Furthermore, under these conditions the ANS emission spectra appeared to have a maximum shifted from the wavelength of 515 nm, observed for free ANS, to that of 480 nm (Fig. 2A). Removal of the aggregates from the sample by centrifugation reduced almost completely the ability of the sample to increase the fluorescence of ANS. Therefore, the observed increase of ANS fluorescence is attributable to the binding of this dye to the aggregates, rather than to partially folded states that could possibly be present in the sample. Congo red spectra were also monitored in the presence of Sso AcP. The protein was pre-incubated for 30 min under the same conditions of temperature and pH and in the presence of 0, 20, 50, and 70% (v/v) TFE. Upon the binding of Congo red to ordered aggregates, the absorption maximum of the dye undergoes a red shift from 490 to ∼540 nm (38Klunk W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1273-1281Google Scholar). The spectra of Congo red obtained in the absence or in the presence of Sso AcP pre-incubated in buffer were highly superimposable, both presenting a maximum at 490 nm (Fig. 3A). By contrast, the spectrum obtained in the presence of Sso AcP, pre-incubated in 20% TFE, had an optical absorption higher than the control spectrum recorded in the absence of protein, throughout the whole wavelength range (Fig. 3B). This originated from the considerable light scattering produced by the protein that yielded an apparently increasing optical absorption as the wavelength decreased (Fig. 3B). The difference spectrum at 20% TFE, obtained by subtracting the spectra of the protein alone and of Congo red alone from the spectrum of the protein in the presence of Congo red, exhibited a maximum at ∼550 nm (Fig. 3C). No apparent maxima were obtained in the difference spectra at the remaining concentrations of TFE (Fig. 3C). Overall, the analysis carried out with ThT, Congo red, and ANS indicate the presence of ordered aggregates within the range 15-25% (v/v) TFE. Less ordered aggregated species seem to be present at higher TFE concentrations, whereas no aggregation seems to occur significantly at concentrations of TFE lower than 15% (v/v), at least on the time scale investigated here. Aggregate Formation Monitored by Dynamic Light Scattering—The evaluation of the presence and size of Sso AcP aggregates was achieved by a DLS analysis. The protein was incubated in various TFE concentrations ranging from 0 to 70% (v/v) for 30 min at 25 °C, pH 5.5. Fig. 4 shows the size distribution by intensity recorded for two representative samples pre-incubated in 0 and 20% (v/v) TFE. Aggregates with an apparent diameter of ∼200 nm were present in 20% TFE (Fig. 4, dotted line). On the contrary, a peak at ∼3.4 nm was present in the protein samples in 0% TFE along with other larger particles (Fig. 4, solid line). The 3.4 nm diameter is highly consistent with that determined for proteins of the acylphosphatase superfamily using NMR or x-ray crystallography (22Pastore A. Saudek V. Ramponi G. Williams R.J. J. Mol. Biol. 1992; 224: 427-440Google Scholar, 23Thunnissen M.M. Taddei N. Liguri G. Ramponi G. Nordlund P. Structure. 1997; 5: 69-79Google Scholar, 24Rosano C. Zuccotti S. Bucciantini M. Stefani M. Ramponi G. Bolognesi M. J. Mol. Biol. 2002; 321: 785-796Google Scholar), suggesting that this peak arises from the native monomer. Since the intensity of the scattered light is proportional to the si
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