In Vitro Formation of Amyloid from r-Synuclein Is Dominated by Reactions at Hydrophobic Interfaces Jeremy Pronchik, Xianglan He, Jason T. Giurleo, and David S. Talaga* Department of Chemistry and Chemical Biology, Wright-Rieman Laboratories, Rutgers, the State UniVersity of New Jersey, New Brunswick, 610 Taylor Road, Piscataway, New Jersey 08854 Received April 6, 2010 E-mail: talaga@rutgers.edu Abstract: Most in vitro investigations of R-Synuclein (RSyn) aggregation and amyloidogenesis use agitation in the presence of air and/or Teflon to accelerate kinetics. The effect of the agitation is implicitly or explicitly attributed to mass transfer or fibril fragmentation. This paper evaluates these hypotheses by agitating RSyn under typical amyloidogenic conditions with controlled numbers of balls made of polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), and borosilicate glass with no headspace. Amyloid was assayed using thioflavin T fluorescence and atomic force microscopy. The observed kinetics were proportional to the PTFE surface area the effects of PMMA and glass balls were negligible by comparison. No amyloid was observed to form in the absence of mixing balls. Agitation with only air also showed accelerated kinetics but different aggregate morphology. The results indicate that the mechanism active in agitation experiments is dominated by reactions at the hydrophobic-water interface. Of the mass transfer, fragmentation, and hydrophobic interface hypotheses, only the last is capable of explaining the data. Condition and sequence determinants of amyloidogenic propensity that have thus far been reported must be reinterpreted as being reflective of partitioning to hydrophobic-water interfaces. Comparable hydrophobic interfaces are not found in vivo. Introduction Parkinson���s disease (PD) is a neurodegenerative disease affecting 1% of the population over 65 and 4-5% over 85.1 R-Synuclein is present in intracellular inclusions that stain positive for amyloid these Lewy Bodies are hallmarks of PD.2 Mutations and triplication of R-Synuclein lead to early onset forms of PD, though sporadic PD accounts for 95% of cases.2 R-Synuclein (RSyn) is a 140 residue, 14.5 kDa protein found in neuronal cells especially in presynaptic termini.2-4 It is an intrinsically disordered protein with instability in its secondary and tertiary structure.5-9 Residues 61-95 of RSyn were first identified as a non-A component (NAC) of amyloid plaques in Alzheimer���s disease.2 The NAC is mostly hydrophobic, but has one positive and one negative residue in close proximity to each other. In experiments where samples are shaken (large air-water interface), RSyn forms amyloid within a few days where RSyn without the NAC does not within 6 weeks.10 Furthermore, the NAC alone will form seeding-competent amyloid.10 RSyn is overall acidic with pI ) 4.6 11 at pH 7.55, the N-terminal region should have 8 negative and 11 positive charges while the C-terminal region has 3 positive and 15 negative charges. In this way, RSyn resembles a triblock copolymer with the hydrophobic NAC lying between charged terminal regions. This structure makes RSyn an excellent surfactant molecule. The link between RSyn and PD has led to intensive in vitro studies of RSyn that aim to determine the aggregation mechanism.2-5,10-21 RSyn amyloidogenesis shows sigmoidal kinetics that can be seeded, consistent with a nucleated process.19,22,23 However, the lag time in the sigmoidal kinetic profile varies by 2 orders of magnitude depending on details of the incubation conditions.3,4,16,21,24 The method used to remove pre-existing aggregates and the use of agitation both have a large impact on the kinetics.15,25,22 Preformed aggregates, such as those created during lyophilization, can reduce the lag time from 30 days to 3 days. In samples without preformed aggregates, shaking with air headspace reduces the lag time from 30 days to several hours.25 Agitating aqueous solutions of RSyn with air or polytetrafluoroethylene (PTFE) leads to faster formation of globular aggregates and amyloid fibrils than without.3,4,16,21,24,25 Most RSyn in vitro experiments use shaking and/or PTFE stirrers to accelerate RSyn amyloid kinetics.3,4,14-16,18-24,26,27 (1) Farrer, M. J. Nat. ReV. Genet. 2006, 7, 306���318. (2) Dauer, W. Przedborski, S. Neuron 2003, 39, 889���909. (3) Conway, K. A. Harper, J. D. Lansbury, P. T. Biochemistry 2000, 39, 2552���2563. (4) Conway, K. A. Lee, S. J. Rochet, J. C. Ding, T. T. Williamson, R. E. Lansbury, P. T. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 571��� 576. (5) Ferreon, A. C. M. Gambin, Y. Lemke, E. A. Deniz, A. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5645���5650. (6) Dunker, A. K. et al. J. Mol. Graphics Modell. 2001, 19, 26���59. (7) Uversky, V. N. Oldfield, C. J. Dunker, A. K. Annu. ReV. Biophys. 2008, 37, 215���246. (8) Eliezer, D. Kutluay, E. Bussell, R. Browne, G. J. Mol. Biol. 2001, 307, 1061���1073. (9) Weinreb, P. H. Zhen, W. Poon, A. W. Conway, K. A. Peter, T. Lansbury, J. Biochemistry 1996, 35, 13709���13715. (10) Giasson, B. I. Murray, I. V. Trojanowski, J. Q. Lee, V. M. J. Biol. Chem. 2001, 276, 2380���2386. (11) Sharon, R. Bar-Joseph, I. Frosch, M. P. Walsh, D. M. Hamilton, J. A. Selkoe, D. J. Neuron 2003, 37, 583���595. Published on Web 06/25/2010 10.1021/ja102896h ��� 2010 American Chemical Society J. AM. CHEM. SOC. 2010, 132, 9797���9803 9 9797

The effect of agitation has been attributed to mixing23,26 and fibril fragmentation.28,29 The mixing hypothesis suggests that aggregation reactions between fibrils and additive species are limited by mass transfer. The mass transfer rates and therefore reaction rates are proposed to be related to the rate of mixing. The fragmentation hypothesis suggests that fibrillization is limited by the number of active nucleation sites. The effect of agitation is to increase sheer forces, which break fibrils, creating more active sites. Both models assume agitation affects reaction steps that occur in homogeneous solution. Both models predict that the kinetics should be independent of the material or interfacial properties of the mixers. An alternative hypothesis is that the dielectric interface created by bubbles and stir bars provides a driving force for the preferential partitioning of protein to the inhomogeneous interface. At the hydrophobic-hydrophilic interface, RSyn is expected to behave like a surfactant, thereby accelerating aggregation reactions by concentrating and conformationally orienting reactive protein species. This hypothesis predicts kinetics to be proportional to interfacial surface area. Investigations into other proteins have demonstrated the sensitivity of aggregation reactions to hydrophobic-water interfaces. Insulin aggregation kinetics have been shown to be proportional to the amount of air-water or Teflon-water interface.30 Hydrophobic, but not hydrophilic, bacteriophages are inactivated with air-water-Teflon interfaces.31 PTFE balls were shown to accelerate the formation of amyloid for the small hormone protein, glucagon.32 Hydrophobic nanoparticles were recently shown to nucleate cross- aggregation of 2-micro- globulin in vitro33 and in a model peptide in silico.34 Grazing incidence X-ray scattering showed that A is random coil in bulk solution but adopts a -sheet when aggregated at the air-water interface.35,36 A model peptide, LSFDNSGAITIG- NH2, spontaneously forms -sheet aggregates at an air-water interface.37 This hypothesis implies that the important reaction steps occur at the heterogeneous interface. It further implies that mutations and solution conditions that modulate kinetics report on changes in the affinity of the protein for the interface. Hydrophobic- hydrophilic interfaces like those of air-water and PTFE-water in vitro experiments have not been found in the mammalian brain and do not appear to be biologically relevant. This paper aims to determine the mechanism by which agitation accelerates the kinetics of amyloidogenesis from RSyn. To evaluate the mixing, fragmentation, and interfacial hypoth- eses, RSyn was incubated under gentle agitation with mixing balls of varying number, density, and hydrophobicity. These conditions systematically changed the efficiency of convection, the local shear forces experienced during agitation, and the driving force for amphiphilic partitioning of RSyn to the interface, respectively. Monodisperse balls provide a fixed-area hydrophobic interface allowing quantitative assessment of its influence. Materials for mixing balls in this study are polytet- rafluoroethylene (PTFE), polymethylmethacrylate (PMMA), and borosilicate glass. PTFE is a chemically inert, highly hydro- phobic fluorinated polymer with a large water contact angle PMMA is modestly hydrophilic glass is hydrophilic. Experimental Section Mixing Balls. Grade 1, 1/16 in PTFE 1 mm PMMA balls (Engineering Laboratories, Inc. Oakland, NJ) and 1 mm glass balls (B. Braun Melsungen Melsungen, Germany) were shaken in several changes of buffer to remove dust before use. Balls were produced by milling and lack a releasing agent (oil) coating. AFM shows the balls to be rough with ���200 nm features (see Supporting Information: Figure S1). The radius of PTFE balls is 0.0625 ( 0.001 in., establishing a minimum 3% error on surface area. Expression of rSyn. Escherichia coli BL21 DE3 strain (Invit- rogen, Inc.) was transfected by a plasmid (pT7-7) encoding human wild-type RSyn (gifts from Prof. Jean Baum Rutgers University, Piscataway, NJ). Expression, purification, and lyophilization of RSyn followed published protocols.21 Thioflavin T (ThT). 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