Amyloid hydrogel derived from curly protein fibrils of a-synuclein Ghibom Bhak a , Soonkoo Lee a , Jae Woo Park a , Sunghyun Cho b , Seung R. Paik a, * a School of Chemical and Biological Engineering, College of Engineering, Seoul National University, 599 Gwanak-Ro, Gwanak-Gu, Seoul 151744, Republic of Korea b Department of Computer Science and Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk 790 784, Republic of Korea article info Article history: Received 29 January 2010 Accepted 29 March 2010 Available online 14 May 2010 Keywords: Self-assembly Amyloidogenesis Hydrogel Protein nanofibrils Nanomatrix abstract Elucidation of molecular assembly mechanism of protein-based suprastructure formation is pivotal to develop biomaterials. A single amyloidogenic protein of a-synuclein turned into two morphologically distinctive amyloid fibrils e ‘curly’ (CAF) vs. ‘straight’ (SAF) e depending on its fibrillation processes. Mutually exclusive production of CAF and SAF was achieved with either centrifugal membrane filtration of the preformed oligomeric species of a-synuclein or agitated incubation of its monomeric form, re- presenting amyloidogeneses via double-concerted and nucleation-dependent fibrillation model, respec- tively. Differences in secondary structures of CAF and SAF have been suggested to be responsible for their morphological uniqueness with structural flexibility and mechanical strength. Both polymorphs exerted the self-propagation property, demonstrating that their characteristic morphologies were inherited for two consecutive generations to daughter and granddaughter fibrils through the seed-dependent fibrillation procedure. Accumulation of CAF produced amyloid hydrogel composed of fine nano-scaled three- dimensional protein fibrillar network. The hydrogel made of daughter CAF was demonstrated to be a suitable nanomatrix for enzyme entrapment, which protected the entrapped enzyme of horseradish peroxidase from loss of activity due to multiple catalyses and heat treatment. The nano-scaled fibrillar network of CAF, therefore, could exhibit a full potential to be further applied in the promising areas of nanobiotechnology including tissue engineering, drug delivery, nanofiltration and biosensor development. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Protein-based suprastructure formation is one of key phenomena involved in a diverse range of biological activities from normal cellular biogenesis to pathogenesis of various degenerative disorders. These suprastructures are also suggested to be engineered in vitro to provide biologically compatible materials [1]. Therefore, elucidation of molecular assembly mechanism for the suprastructure formation is crucial not only to understand its implications in biology but also to obtain biomaterials for their eventual applications in the area of nanobiotechnology. Amyloid fibrils are highly ordered fibrillar protein aggregates known to be stabilized predominantly by cross-b-sheet confor- mation [2]. They are the product of disorder-to-order transition of partially misfolded proteins through selective molecular self- assembly process [3]. Mature amyloid fibrils elicit polymorphism resulted from either molecular-level structural variations of indi- vidual amyloidogenic proteins or alterations in intra- or inter- fibrillar interactions [4]. This amyloid formation has been closely associated with neurodegenerative disorders including Parkinson’s disease (PD), Alzheimer’s disease (AD), and Prion disease [5] although toxic cause of the cellular degeneration remains unset- tled [6]. The amyloid fibrils exhibit biological activities such as biofilm formation of enterobacteria, hyphae formation of fungi, egg envelope formation of insects and fish, biosynthesis of melanin within mammalian melanocytes and activation of factor XII in human hemostasis [7,8]. In addition, the amyloid fibrils are also considered as protein nanofibrils with an average width of 10e20 nm and a mechanical strength comparable to spider silk [9]. This nano-scaled biomaterial has been suggested to have a full potential for applications by providing functional templates for conductive nanowire preparation, nanoparticle alignment and enzyme immobilization and by turning into liquid crystal state and hydrogel formation [10]. Exploration of diversified amyloid fibril- lation procedures, therefore, will bring us practical means to obtain various functional nanomaterials for biotechnological applications. Molecular self-assembly mechanism for the amyloidogenesis has been prevalently modeled with the nucleation-dependent fibrillation process [11e 13]. After thermodynamically unfavorable nucleus formation, the fibrillation process is facilitated by accreting monomeric units in either pre-structured or unstructured form to exhibit template complementarity, which has been reflected on the * Corresponding author. Tel.: þ82 2 880 7402; fax: þ82 2 888 1604. E-mail address: srpaik@snu.ac.kr (S.R. Paik). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.03.080 Biomaterials 31 (2010) 5986e5995