Accelerated Publications Aâ Protofibrils Possess a Stable Core Structure Resistant to Hydrogen Exchange†

Protofibrils are transient structures observed during in vitro formation of mature amyloid fibrils and have been implicated as the toxic species responsible for cell dysfunction and neuronal loss in Alzheimer’s disease (AD) and other protein aggregation diseases. To better understand the roles of protofibrils in amyloid assembly and Alzheimer’s disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. We chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Aâ(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We show that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We find that about 40% of the backbone amide hydrogens of Aâ protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also find a surprising degree of specificity in amyloid assembly, in that wild type Aâ is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides. These and other data are interpreted and discussed in terms of the role of protofibrils in fibril assembly and in disease. Amyloid fibrils are the primary protein component of neuritic plaques found in the brains of Alzheimer’s disease (AD)1 patients and are associated with more than 20 other amyloid diseases (3). In AD, the amyloid plaques are composed primarily of the Aâ peptide, a 39-43 residue proteolytic product of a transmembrane protein called amyloid precursor protein (APP) (4). All four types of mutation associated with early onset familial AD are linked to increased production and/or deposition of Aâ peptides in the brain (5). Although some studies find that the classically stained Aâ plaque burden does not correlate perfectly with AD progression (6), there does appear to be a correlation between disease and the ratio of soluble to insoluble Aâ in the brain (7). One attractive hypothesis for resolving the contrasting genetic and pathological trends is that the toxic agent in AD is not the extracellular amyloid plaque but rather another aggregated form of Aâ, possibly a prefibrillar oligomeric assembly intermediate. Nonfibrillar oligomeric forms of Aâ clearly exist. Oligomeric forms collectively referred to as protofibrils are typically transiently observed when relatively high, nonphysiological concentrations of monomeric Aâ are incubated in vitro to generate amyloid fibrils (8, 9). Oligomeric forms of Aâ have also been characterized in cell cultures (10, 11). Exogenous and endogenous Aâ protofibrils are cytotoxic (11, 12), a property they share with oligomeric assembly intermediates of other amyloidogenic proteins (13). Striking evidence in support of a role for protofibrils in AD comes from a recently described early onset familial AD mutation in the Aâ peptide itself (E22G) called the Arctic mutation (14). The Arctic mutation in Aâ (AâARC) has an increased propensity for forming protofibrils as compared to wild type Aâ (AâWT), suggesting that this mutation predisposes individuals to early-onset AD due to the formation of relatively long-lived, toxic protofibrils (14). † Supported in part by NIH R01AG18927 to R.W. and NIH R01AG08470 to P.T.L. * To whom correspondence should be addressed. Phone: (865) 5449168. Fax: (865) 544-9235. E-mail: [email protected]. ‡ University of Tennessee. § These authors have contributed equally to the work presented in this publication. | Brigham and Women’s Hospital and Department of Neurology, Harvard Medical School. ⊥ Department of Cell Biology, Harvard Medical School. 1 Abbreviations: HX-MS, hydrogen-deuterium exchange mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; SEC, size exclusion chromatography; AD, Alzheimer’s disease; PBS, phosphate-buffered saline; Tris, tris(hydroxymethyl)aminomethane; TFA, trifluoroacetic acid; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol. 14092 Biochemistry 2003, 42, 14092-14098 10.1021/bi0357816 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/12/2003 Recently, Lashuel and co-workers (15) identified conditions that permit the preparation, separation (by size exclusion chromatography (SEC)), and biophysical characterization of stable protofibrillar species of Aâ. Under these conditions, electron microscopic (EM) examination of the protofibrils formed by Aâ(1-40)ARC revealed several morphologies, including (a) relatively compact spherical particles roughly 4-5 nm in diameter, (b) annular pore-like protofibrils (6-9 nm o.d. and 1.5-2 i.d.), (c) large spherical particles 18-25 nm in diameter, and (d) short filaments with chainlike morphology. Interestingly, protofibrils formed from an equimolar mixture of AâWT and AâARC, a biologically relevant mixture of the two proteins that may model the situation in heterozygous patients, are more stable than those formed by AâARC and AâWT alone (15). To better understand their roles in both assembly of amyloid fibrils and Aâ cytotoxicity, it is important to further characterize the structural properties of these protofibrils. Due to their large size, heterogeneous morphology, and poor solubility, amyloid fibrils and protofibrils have so far resisted high-resolution structure determination. The metastability of protofibrils presents an additional impediment. There are, however, intermediate resolution methods that can provide insights into the structures of monomers, protofibrils, and fibrils and to the structural relationships among these states. In particular, hydrogen-deuterium exchange (HX) provides significant insight into structure by determining the pattern and extent of hydrogen bonding within the aggregate (1). HX methods take advantage of the fact that exchange of backbone amide protons involved in H-bonded secondary structures such as R-helices and â-sheets, and/or those buried in core structure, is substantially slower in comparison to backbone protons in non-H-bonded regions such as loops and random coil structures. HX has been widely used in conjunction with mass spectrometry (MS) to study protein structure and dynamics (16, 17). HX-MS techniques have recently been extended to study the structures of amyloid fibrils (1, 2, 18, 19). Using these methods, we found that Aâ amyloid fibrils consist of a very rigid core structure, most likely an H-bonded â-structure, which involves only 19 of the 39 Aâ backbone amides (1, 2). In this paper, we extend these methods to characterize the structure in the soluble oligomeric intermediates formed in AâARC and mixed AâARC/AâWT fibrillization reactions. Details of protofibril structure may provide critical insights into the mechanism of amyloid fibril formation and its role in the mechanism of pathogenesis as well as the design and development of AD therapeutics. MATERIALS AND METHODS Protofibril Synthesis. Protofibrils were prepared from chemically synthesized AâWT(1-40) (NH2-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-COOH) and AâARC(1-40 Arctic (E22G)) monomers purchased as trifluoroacetic acid (TFA) salts from the Biopolymer Facility at Brigham and Women’s hospital. The detailed procedure for the preparation of Aâ protofibrils has been described previously (15). Briefly, lyophilized Aâ monomer (either Arctic or an equimolar mixture of wt and Arctic) was dissolved in 1 mM NaOH and then diluted to 100 μM in 0.2 × PBS, pH 7.4 followed by incubation at room temperature for 16-24 h. Large, insoluble particles (consisting of 15-20% of the total Aâ monomer) were removed by 5 min centrifugation at 13 000g and hereafter will be referred to as pellet. Protofibrils and small soluble Aâ species in the supernatant were separated using a Superdex 75 HR (Amersham Pharmacia) SEC column equilibrated with 5 mM Tris-HCl, pH 7.4, 70 mM NaCl. The protein was eluted at a flow rate of 0.5 mL/min. The size exclusion chromatogram consisted of a void volume peak corresponding to oligomeric species of different sizes and morphologies (protofibrils) and a relatively sharp peak corresponding to species of smaller hydrodynamic radii, predominantly monomeric Aâ (low molecular weight; LMW) (15). The presence of small amounts of rapidly equilibrating oligomers (dimers, trimers, and tetramers) in the LMW peak cannot be ruled out. The oligomeric and LMW peaks were split into several fractions in the order of their elution (PF1-PF3 and LMW1 and LMW2) and stored at 4 °C. The pellet and the protofibril fractions were spotted on the grids for EM and also expressshipped on ice for HX experiments. Hydrogen Exchange. SEC fractions were stored on ice after their isolation, and HX experiments were initiated within 24 h and completed within another 48 h. A 250-500 μL aliquot of protofibril fractions was placed in a YM-10 microcon tube (Millipore) and centrifuged at 14 000g at 3 °C. Since highly concentrated protofibril samples readily form fibrils, the centrifugation time (30-50 min) was selected to prevent their concentration on the filter by more than 5-fold. Each concentrated sample was diluted with D2O and centrifuged at 3 °C, and the process was repeated one to two times until the H2O content reached <2%. The filter was then inverted and centrifuged at 1000g for 3 min to collect the sample in a fresh centrifuge tube, and the sample was analyzed for deuterium content after being adjusted to an equivalent monomer concentration of 15-20 μM. The average time to prepare each sample was 2.5 ( 0.5 h. This method of HX sample preparation maintained the integrity of the protofibril sample while removing salts and introducing D2O. After HX analysis, samples were held on ice for up to an additional 24 h and then were analyzed by EM. Importantly, EMs of the HX analyzed samples confirmed that the protofibril fractions had not progressed to mature fibrils during analysis (see Results and Figure 1). The pellets formed during the preparation of AâARC protofibrils (after 16 h of incubation at 37 °C) were either incubated at 37 °C for an additional 24 h to generate mature fibrils or were stored on ice until HX experiments were carried out. Pure AâWT (1-40) fibrils were generated by disaggregating monomer using TFA and hexafluoroisopropanol (HFIP) (Pierce) followed by incubation at 37 °C in 1X PBS for 5-7 days (20). For HX experiments on fibrils and pellets, aggregates were collected by centrifugation, washed once with 2 mM TrisHCl buffer, and resuspended in D-Tris-DCl buffer as described in detail previously (1). For HX experiments on monomers, AâARC and AâWT were disaggregated in TFA and HFIP as stated previously, lyophilized, then dissolved in 2 mM D-Tris-DCl buffer, and immediately analyzed as described previously (1). ESI-MS. The HX equipment and protocol have been described previously (1, 2). Briefly, the Z electrospray source of a Quattro II (Micromass) triple quadrupole mass specAccelerated Publications Biochemistry, Vol. 42, No. 48, 2003 14093