Aspen SP1, An Exceptional Thermal, Protease and Detergent-Resistant Self-Assembled Nano-Particle Wang-Xia Wang, 1,3 Or Dgany, 1 Sharon Grayer Wolf, 2 Ilan Levy, 1 Rachel Algom, 1 Yehonathan Pouny, 3 Amnon Wolf, 3 Ira Marton, 3 Arie Altman, 1 Oded Shoseyov 1 1 The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, and the Otto Warburg Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel; telephone: 972-8-9489084; fax: 972-8-9462283; e-mail: shoseyov@agri.huji.ac.il 2 Electron Microscopy Unit, Weizmann Institute of Science, Rehovot 76100, Israel 3 Fulcrum SP Ltd., P.O.Box 3206, Herzliya Pituach 46104, Israel Received 10 January 2006; accepted 5 April 2006 Published online 26 May 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.21010 Abstract: Stable protein 1 (SP1) is a homo-oligomeric protein isolated from aspen (Populus tremula aspen) plants which forms a ring-shape dodecameric particle with a central cavity. The oligomeric form of SP1 is an exceptionally stable structure that is resistant to proteases (e.g., trypsin, V8, and proteinase K), high temperatures, organic solvents, and high levels of ionic detergent. Analytical ultra-centrifugation, chemical cross-linking, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and transmission elec- tron microscopy were used to further characterize the SP1 dodecamer. Introduction of a single cysteine at the N-terminus of SP1 enabled the formation of disulfide bridges within the SP1 dodecamer, concurrent with increased melting point. A six-histidine tag was intro- duced at the N-terminus of SP1 to generate 6HSP1, and the DNSP1 mutant was generated by a deletion of amino acids 2–6 at the N-terminus. Both 6HSP1 and DNSP1 main- tained their ability to assemble a stable dodecamer. Remarkably, these SP1 homo-dodecamers were able to re-assemble into stable hetero-dodecamers following co-electro-elution from SDS–PAGE. The exceptional stability of the SP1-nano ring and its ability to self- assemble hetero-complexes paves the way to further research in utilizing this unique protein in nano-biotech- nology. ß 2006 Wiley Periodicals, Inc. Keywords: tremula; oligomer; stable; self-assembly; nanoparticle INTRODUCTION Stable protein 1 (SP1), isolated from aspen plants (Populus tremula), responds to a wide range of environmental stresses, including salinity, cold, and heat stress, and accumulates during stress recovery (Wang et al., 2002). SP1 forms a ring- shaped homo-dodecamer that does not precipitate upon boiling (Dgany et al., 2004; Wang et al., 2002). Crystal structure studies showed that SP1 belongs to the aþb protein class with a ferredoxin-like fold composed of a aþ b-sandwich with an anti-parallel sheet (Dgany et al., 2004). One of the proteins bearing such a ferredoxin-like fold structure, TT1380, was isolated from a thermophilic bacterium, Thermus thermophilus (Wada et al., 2004). This may imply that the two proteins share a structural basic scaffold which confers thermostability. Indeed, a differential scanning calorimetric study showed that SP1 has high thermostability, that is, Tm of 1078C (at pH 7.8) (Dgany et al., 2004). Although no significant sequence similarity has been found with known protein families, SP1 homologs have been observed in a number of plant and bacterial species, either as putative proteins from genomic sequences or ESTs with unknown function (Wang et al., 2002). Nanotechnology is a fast emerging field of science and industry. One of the fundamentals of nanotechnology is self- assembly. Self-assembly describes the spontaneous organi- zation of molecules into structurally defined arrangements through a number of non-covalent interactions (Lehn, 1993; Whitesides et al., 1991). These molecules reassemble through hydrogen bonds, ionic bonds, and van der Waal’s bonds, forming hierarchical structures. Although each of the bonds is rather weak, the accumulation of many interactions can result in stable structures and materials. Chemical and structural compatibility are the key elements in self- assembly. These elements are reflected in coded information: shape, surface properties, charge, and polarizability (White- sides, 2002). Viruses are one of the most intriguing examples ß 2006 Wiley Periodicals, Inc. Correspondence to: Oded Shoseyov Wang-Xia Wang and Or Dgany are equal contributors. Wang-Xia Wang’s present address is Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.