Notes & Tips Ionic liquids promote amyloid formation from a-synuclein Heejin Hwang 1 , Hyunghun Choi 1 , Hyun-kyung Kim 2 , Do Hyun Jo, T. Doohun Kim * Department of Molecular Science and Technology, Graduate School of Interdisciplinary Program, Ajou University, Suwon 443-749, Republic of Korea article info Article history: Received 6 November 2008 Available online 24 December 2008 abstract The slow process required for a-synuclein to form amyloid fibrils is a major obstacle in the development of therapeutic compounds for a-synuclein-related neurodegenerative diseases such as Parkinson’s dis- ease (PD). Here we have developed an efficient method by which amyloid fibrils can be formed from a-synuclein using ionic liquids (ILs). This report indicates that ILs could potentially be used as a stimu- lator for the amyloid formation of a-synuclein. Ó 2008 Elsevier Inc. All rights reserved. a-Synuclein is a small (14 kDa) cytoplasmic protein that is unstructured and highly soluble at presynaptic terminals in the neurons. Although the physiological role of a-synuclein is not clearly understood, it has been implicated for its involvement in synaptic plasticity, neurotransmitter release, and cellular traffick- ing in the cell [1,2]. The primary sequences of a-synuclein are com- posed of three regions: an N-terminal region with KTKEGV repeats, a central hydrophobic region, and a C-terminal region with acidic residues. In previous studies, the central region (amino acids 61– 95) has been shown to play a key role in the formation of amyloid fibrils, which is a critical step in the pathogenesis of a-synuclein- related neurodegenerative diseases such as Parkinson’s disease (PD) 3 [3]. Recombinant a-synuclein could form amyloid fibrils through a nucleation-dependent mechanism, and it undergoes con- formational changes from a highly unstructured state to b-strands- rich amyloid fibrils through neurotoxic intermediates [4,5]. To devel- op an effective therapeutic strategy for amyloid-related diseases, the amyloid formation process must be investigated at molecular level both in vivo and in vitro. However, a-synuclein generally takes sev- eral weeks or even months to form amyloid fibrils, although this pro- cess can be accelerated by increased temperature, acidic pH, C- terminus truncation, and high concentrations of organic compounds [6–8]. Therefore, this slow a-synuclein-related process is a major obstacle to the development of therapeutic compounds to regulate the amyloid formation through the a-synuclein process. Here we have developed an efficient method for regulating the formation of amyloid fibrils using ionic liquids (ILs). ILs are organic salts with high polarity, negligible vapor pressure, and thermal stability [9]. The cat- ionic parts of ILs are usually made of imidazolium, N-alkylpyridini- um, tetraalkylammonium, or tetraalkylphosphonium, whereas the anionic parts are halides, nitrate, acetate, hexafluorophosphate ([PF 6 ]), and tetrafluoroborate ([BF 4 ]). We hypothesized that because ILs could form extensive electrostatic interactions in solution, they could effectively drive the assembly or aggregation of amyloid-prone proteins such as a-synuclein. Human a-synuclein was highly purified and filtered through a 0.22-lm filter prior to use [3,10]. a-Synuclein was incubated with 13 different ILs of similar molecular weights (MWs) but diverse chemical groups (see Table 1 in Supplementary material). We used thioflavin T (ThT) assays to confirm the amyloid aggregates and transmission electron microscopy (TEM) to create images of the morphology of the fibrils. ThT shows enhanced fluorescence at 490 nm when bound to amyloid fibrils but not native or amorphous aggregates. Assays were performed in a total volume of 200 ll of 100 lM ThT in phosphate buffer (50 mM Tris–phosphate and 150 mM NaCl [pH 8.0]) with 1 mg/ml a-synuclein. The fluorescence intensities of samples and a blank containing only 100 lM ThT were compared by measuring intensities in a Jasco FP-6200 spectrofluo- rometer by excitation at 450 nm (5 nm slit width) and emission at 490 nm (5 nm slit width). Data were collected at 24-h intervals with a 15-s integration time. A negative control (1 mg/ml a-synuc- lein in phosphate buffer) was included to establish background lev- els of fluorescence due to nonspecific binding of ThT. Fig. 1 shows the fibril formation of a-synuclein as monitored by ThT fluorescence. To address the role of ILs in amyloid formation, we chose to examine a series of ILs with similar MWs. This series of ILs allowed us to determine the properties that are important for the stability and kinetics of amyloid formation of a-synuclein. As shown in the upper panel of Fig. 1, the introduction of ILs sub- stantially decreased the lag time of amyloid formation of a-synuc- lein, indicating that ILs enhance nuclei formation. The addition of ILs is critical because moderate agitation for several weeks or even 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.12.019 * Corresponding author. Fax: +82 31 219 2394. E-mail address: doohunkim@ajou.ac.kr (T.D. Kim). 1 These authors contributed equally to this work. 2 Present address: Korea Institute for Curriculum and Evaluation. 3 Abbreviations used: PD, Parkinson’s disease; IL, ionic liquid; [PF 6 ], hexafluoro- phosphate; [BF 4 ], tetrafluoroborate; MW, molecular weight; ThT, thioflavin T; TEM, transmission electron microscopy. Analytical Biochemistry 386 (2009) 293–295 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio