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7828.Full.Pdf Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation Nata` lia Carullaa,1, Min Zhoub, Muriel Arimonc, Margarida Gairíd, Ernest Giralte,f, Carol V. Robinsonb,1, and Christopher M. Dobsonb,1 aInstitucio´Catalana de Recerca i Estudis Avanc¸ats Researcher at Institut de Recerca Biome`dica, Baldiri Reixac 10-12, 08028 Barcelona, Spain; bDepartment of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB21EW, United Kingdom; cInstitut de Bioenginyeria de Catalunya, Baldiri Reixac, 15, 08028 Barcelona, Spain; dUnitat de RMN, Serveis de Suport a la Recerca, Universitat de Barcelona, Baldiri Reixac, 10-12, 08028 Barcelona, Spain; eInstitut de Recerca Biome`dica, Baldiri Reixac 10-12, 08028 Barcelona, Spain; and fDepartament de Química Orga`nica, Universitat de Barcelona, Martí i Franque`s 1, Barcelona 08028, Spain Edited by David Baker, University of Washington, Seattle, WA, and approved March 10, 2009 (received for review December 2, 2008) Recent experimental evidence points to intermediates populated forms of mass spectrometry (12, 13). In addition, a conformation- during the process of amyloid fibril formation as the toxic moieties specific antibody that recognizes soluble oligomers from many types primarily responsible for the development of increasingly common of proteins, regardless of sequence, has been produced and has disorders such as Alzheimer’s disease and type II diabetes. We proved to be very useful in monitoring the kinetics of oligomer describe here the application of a pulse-labeling hydrogen- formation (14). deuterium (HD) exchange strategy monitored by mass spectrom- HD exchange experiments are based on solvent accessibilities; etry (MS) and NMR spectroscopy (NMR) to characterize the aggre- amide protons that normally undergo rapid exchange with solvent gation process of an SH3 domain under 2 different conditions, both deuterons experience much slower exchange when involved in of which ultimately lead to well-defined amyloid fibrils. Under one H-bonded structures and/or when sterically inaccessible to the condition, the intermediates appear to be largely amorphous in solvent. HD exchange experiments can be applied to probe systems nature, whereas under the other condition protofibrillar species either at equilibrium or during the kinetic chain of reactions are clearly evident. Under the conditions favoring amorphous-like following perturbation from equilibrium conditions. In equilibrium intermediates, only species having no protection against HD ex- experiments, the protein conformation under study does not change can be detected in addition to the mature fibrils that show change with time. In the context of protein aggregation, equilibrium a high degree of protection. By contrast, under the conditions HD exchange experiments have been used to probe the core favoring protofibrillar-like intermediates, MS reveals that multiple structure of a range of amyloid fibrils (8–10) and protofibrils (15) species are present with different degrees of HD exchange pro- and the dynamics of molecular recycling within an ensemble of tection, indicating that aggregation occurs initially through rela- fibrils (16). By contrast to such equilibrium studies, kinetic HD tively disordered species that subsequently evolve to form ordered exchange experiments can monitor protein conformational changes as a function of time. Within this context, we have developed a aggregates that eventually lead to amyloid fibrils. Further analysis pulse-labeling HD exchange experiment designed to gain both using NMR provides residue-specific information on the structural mechanistic information on the process of aggregation and struc- reorganizations that take place during aggregation, as well as on tural information on the different species present during the course the time scales by which they occur. of aggregation (Fig. 1). In this article, we describe the application of such pulse-labeling ͉ ͉ ͉ aggregation HD exchange misfolding intermediates PI3-SH3 HD exchange experiments to the study of the aggregation process of the SH3 domain of the ␣-subunit of bovine phosphatidylinositol- rotein aggregation into amyloid fibrils is associated with a wide 3Ј-kinase (PI3-SH3). The PI3-SH3 domain is a protein that aggre- Prange of increasingly prevalent disorders such as Alzheimer’s gates to form well-characterized fibrils in vitro, particularly at low disease, Creutzfeldt–Jakob disease, and type II diabetes (1, 2). pH (17, 18). Moreover, these fibrils and their precursors show Amyloid fibril formation appears to be a multistep process during structural and cytotoxic properties that are closely similar to those which a series of intermediate aggregated states is sampled (3). observed in many depositional disorders (19). The results described Although early hypotheses proposed amyloid fibrils as the primary in this article provide important information about the aggregation pathogenic agent, much recent evidence supports the view that process of a protein, such as the distribution and stability of the smaller aggregates populated during the fibril assembly process different aggregation states and the nature of structural reorgani- represent the primary culprits (4, 5). The importance of the details zations occurring during amyloid fibril formation. of the aggregation process to the mechanism of disease makes characterization of the different species present during aggregation Results an issue of extreme importance. Of all of the species formed during A Pulse-Labeling Strategy for HD Exchange Analysis of Aggregation aggregation, amyloid fibrils, despite their size and apparent intrac- Intermediates. The pulse-labeling HD exchange experiment de- tability, are the easiest to characterize because of their long-lived signed to probe the species present at different stages in the nature and great regularity. Application of solid-state NMR (NMR) techniques (6), site-directed spin labeling in combination Author contributions: N.C., M.Z., E.G., C.V.R., and C.M.D. designed research; N.C., M.Z., and with electron paramagnetic resonance (EPR) (7), and hydrogen M.A. performed research; M.G. contributed new reagents/analytic tools; N.C., M.Z., and deuterium (HD) exchange experiments (8–10) have all made very M.A. analyzed data; and N.C., M.Z., E.G., C.V.R., and C.M.D. wrote the paper. significant contributions to the study of their structures. Prefibrillar The authors declare no conflict of interest. intermediates, however, are very difficult to characterize because This article is a PNAS Direct Submission. they are short-lived, are often heterogeneous, and may be present 1To whom correspondence may be addressed. E-mail: [email protected], at low populations. Despite these problems, several techniques have [email protected], or [email protected]. been developed to study such species, notably photo-induced This article contains supporting information online at www.pnas.org/cgi/content/full/ cross-linking of unmodified proteins (PICUP) (11) and various 0812227106/DCSupplemental. 7828–7833 ͉ PNAS ͉ May 12, 2009 ͉ vol. 106 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812227106 Downloaded by guest on September 29, 2021 ∆ ∆ A tagg tlabel A 2 d 6 d 21 d PI3-SH3 in D2O 1. Quench by freeze-drying aggregating conditions Aggregating buffer 2. Dissolution of the aggregates in H2O 3. MS and NMR analysis Buffer exchange 1. Freeze-drying B D D from D2O to H2O H H 2. Dissolution of the D H ∆ D D Dialysis or Dilution H H aggregates short tagg D H D ∆ H 3. MS and NMR in D2O D tlabel D 0 d 3 d 21 d D D D H H D D D D D analysis H H B D D D D D H D D D H H D D D Ordered H H H H D Aggregate H H H H D D H H H D D H H H D Soluble protein H D H 1. Freeze-drying D D D D 2. Dissolution of the C D D Buffer exchange H D D H aggregates D D from D2O to H2O ∆ D D 3. MS and NMR long tagg D D Dialysis or Dilution H D D H D 0123456789 ∆ analysis D H C D in D2O D D tlabel D D D D H D D D H D D D H D D D H 10 11 12 13 14 15 17 19 21 C D D H H D D H H D D H Fibril H D H D D H H D H D Soluble protein H Fig. 2. Characterization of the PI3-SH3 aggregation process under AM conditions. (A) Electron micrographs obtained for ⌬tagg of 2, 6, and 21 days. Fig. 1. Schematic description of the pulse-labeling HD exchange experiment (Scale bar, 200 nm.) (B) AFM images obtained for ⌬tagg of 0, 3, and 21 days. developed to study protein aggregation. (A) The experiment starts by incu- (Scale bar, 100 nm.) (C) Kinetics of oligomer-specific immunoreactivity. At the bating soluble protein under aggregation conditions in a deuterium based times indicated, aliquots were applied to a nitrocellulose membrane and ⌬ buffer. After a variable aggregation time, tagg, labeling takes place for a probed with the oligomer-specific antibody, A11. The spot marked with a C ⌬ fixed period, tlabel, using protonated aggregation buffer. The magnitude of corresponds to A␤ oligomers and was used as a positive control. ⌬tlabel is chosen so that only unprotected amide deuterons will exchange significantly with the solvent. After the labeling pulse, freeze-drying is used to ⌬ quench exchange. Different samples are prepared at defined tagg values, coexist with fibrillar ones and from 10 days onward, electron which are later solubilized into monomers by transfer to a DMSO solution and micrographs reveal the presence of highly abundant and morpho- analyzed by NMR and ESI-MS. The figure illustrates hypothetical scenarios logically well-defined fibrils (Fig. 2A). Similar types of aggregates when the protein is left to aggregate for a short ⌬tagg (B) and a long ⌬tagg (C). are observed by AFM (Fig. 2B), except for the amorphous ones, such aggregates may not adhere well to the graphite surface or they aggregation reaction involves first the incubation of soluble protein may be missed during imaging because of low tip adhesion.
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