proteins STRUCTURE O FUNCTION O BIOINFORMATICS Distinct phases of free a-synuclein—A Monte Carlo study Sigurður Ægir Jo ´ nsson, 1 Sandipan Mohanty, 2 and Anders Irba ¨ck 1 * 1 Computational Biology & Biological Physics, Department of Astronomy and Theoretical Physics, Lund University, So ¨lvegatan 14A, SE-223 62 Lund, Sweden 2 Institute for Advanced Simulation, Ju ¨lich Supercomputing Centre, Forschungszentrum Ju ¨lich, D-52425 Ju ¨lich, Germany INTRODUCTION Accumulation of a-synuclein (aS) is widely held to play a role in the pathogenesis of Parkinson’s disease (PD). 1,2 Aggregates of aS occur in Lewy bodies and Lewy neurites, 3,4,5 the intraneuronal hallmarks of PD, and a group of related disorders (synucleinopathies). Over the past two decades, a great deal has been learned about different forms assumed by aS. It was shown that aS aggregates into b-sheet-containing amy- loid fibrils, 6 and a fibril model was developed. 7–10 Although less is known about aS oligomers, progress was made in this area as well. 11 Membrane-bound forms of aS were identified and found to be helical. 12–14 Recent evidence suggests that the main physiological form of aS is a folded helical tetramer. 15 The ability of aS to assume these different forms brings into focus the properties of the free monomer. The 140- residue aS molecule is composed of three parts with phys- icochemically different characteristics: the N-terminal 1–60 region (N), the ‘‘non-amyloid b component’’ region com- prising residues 61–95 (NAC), 16 and the C-terminal 96– 140 region (C). At neutral pH, the N, NAC, and C regions carry net charges of 14, 21, and 29, respectively. The N region contains many charged residues, despite a modest net charge. The NAC region has many hydrophobic resi- dues and few charged ones, and is believed to play a driv- ing role in aS aggregation. 17 Experimental studies of the free aS monomer have demonstrated that it is disordered 18,19 and not much smaller in extension than a random coil. 20 The topology of this state was examined by NMR techniques such as paramagnetic relaxation enhancement (PRE) and residual dipolar couplings. 21–25 Early studies found long-range contacts between the C region and the rest of the mole- cule to be more frequent than expected for a random coil, 21,22 and it was proposed that such contacts slow down aggregation by making the hydrophobic NAC region less solvent exposed. Recently, this hypothesis has been questioned. 23–25 It was found that neither reduc- tion of pH, which accelerates aggregation, 26 nor aggrega- tion enhancing mutations led to an increase of the sol- vent exposure of the NAC region. Although the disordered state of free aS is well estab- lished by a variety of experiments, its detailed properties Grant sponsor: Swedish National Infrastructure for Computing; Grant number: SNIC 001/11-130; Grant sponsor: Ju ¨lich Supercomputing Centre; Grant number: JJSC 03. *Correspondence to: A. Irba ¨ck, Computational Biology & Biological Physics, So ¨lvegatan 14A, SE-223 62 Lund, Sweden. E-mail: anders@thep.lu.se. Received 27 February 2012; Revised 16 April 2012; Accepted 25 April 2012 Published online 2 May 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.24107 ABSTRACT The a-synuclein protein (aS), implicated in Parkinson’s disease, shows conformational versatility. It aggregates into b-sheet- rich fibrils, occurs in helical membrane-bound forms, is disordered as a free monomer, and has recently been suggested to have a folded helical tetramer as its main physiological form. Here, we use implicit solvent all-atom Monte Carlo methods to explore the conformational ensemble sampled by the free aS monomer. We analyze secondary structure propensities, size, and topological properties and compare with existing experimental data. Our study suggests that free aS has two distinct phases. One phase has the expected disordered character. The other phase also shows large conformational variability. How- ever, in this phase, the b-strand content is substantial, and the backbone fold shows statistical similarities with that in aS fibrils. Presence of this phase is consistent with data from low-temperature experiments. Conversion of disordered aS to this fibril-like form requires the crossing of a rather large apparent free-energy barrier. Proteins 2012; 00:000–000. V V C 2012 Wiley Periodicals, Inc. Key words: protein folding; protein misfolding; protein aggregation; amyloid; conformational heterogeneity. V V C 2012 WILEY PERIODICALS, INC. PROTEINS 1