1 Exploring Protein Amyloids in Synthetic Biology Amyloid Assemblies: Protein Legos at a Crossroads in Bottom-Up Synthetic Biology Rafael Giraldo* [a] One of the major objectives that bottom-up Synthetic Biology shares with Chemical Biology is to engineer extant biological molecules to implement novel functionalities in living systems. Proteins, due to their astonishing structural and functional versatility and to their central roles in the biology of cells, should be cornerstones of Synthetic Biology. In particular, protein amyloid cross-β assemblies constitute one of the most stable, conceptually simple, and universal macromolecular architectures ever found in Nature and thus have enormous potential to be explored. This article focuses on the concepts behind the usage of the amyloid cross-β structural framework as a Synthetic Biology part, underlining recent basic findings and ideas. The pros and the cons associated with the polymorphism and the cellular toxicity of protein amyloids are also discussed, keeping in mind the possible suitability of these protein assemblies for scaffolding novel orthogonal macromolecular devices in vivo. Introduction Protein amyloids have a common, simple yet extremely stable, three-dimensional (3D) structure termed cross-β: multiple copies of a peptide stretch, each provided by a different molecule of the same protein, assemble as strands in β-sheets of indefinite extension which then stack and twist as fibres. [1] Main chain hydrogen-bonding between residues in the individual adjacent strands (spaced by ~4.7 Å apart), together with the chemical properties of the involved side chains, determine their parallel or antiparallel arrangement in the β-sheets. A feature of cross-β amyloids is that packing of contiguous sheets (spaced by ~9-11 Å apart) proceeds through interdigitation of compatible side chains (either hydrophobic or polar) in a sort of steric zipper. [2] Since conditions leading to amyloid aggregation can be found for many different proteins, the amyloid state can be regarded as a sort of primordial or ancestral protein fold. [1] Conceptually, amyloid cross-β fibres share some of the formal beauty of DNA double helices, the icon of Molecular Biology. It is thus not strange that amyloids hold an irresistible attraction for protein scientists. But interest in amyloids goes far beyond Protein Science, since they are the causative agents of a number of serious, often fatal, human neurodegenerative and systemic diseases, including those caused by infectious protein particles (aka prions), collectively termed amyloid proteinopathies. [3] These have made amyloids the subject of intense biomedical research in the last two decades, with a focus on the quest for small molecules inhibiting amyloidogenesis. [4] In addition, the aggregated amyloid states of an increasing number of proteins are being reported to have physiological roles, including CsgA-curlin (paving the extracellular lattice which holds bacterial biofilms), Pmel17 (a scaffolding protein for enzymes in the synthesis of melanin in vertebrates) and CPEB (involved in memory storage in the mollusk Aplysia). [5] To the same class of functional amyloids belong yeast prions, with their enormous adaptative potential as epigenetic determinants. [6] The ubiquitous presence of protein amyloid assemblies in Nature justifies placing them at the core of the emergent realm of Synthetic Biology. Synthetic Biology has many different definitions, [7] in part due to its transdisciplinary character, but the so-called bottom-up approach to Synthetic Biology is closely aligned with the interests of Chemical Biology: Synthetic Biology aims to design modules and devices, based either on natural or artificially modified macromolecular parts, enabling novel functionalities in reconstructed biological systems, or in those built de novo. [7] The astonishing versatility and properties of protein amyloids as macromolecular assemblies place them at a crossroads in Synthetic Biology (Scheme 1). Knowledge of the biophysical (structural, thermodynamic and kinetic) basis for amyloid assembly is progressing at an astonishing pace. Recent basic research efforts have shed light on issues such as polymorphism [8] and the characterization of amyloidogenic effectors. [9] Besides this physicochemical interest, amyloids can be valuable tools in a number of established and emergent fields. In Biotechnology, amyloids can be used to engineer ligand- modulated irreversible switches in macromolecular assemblies, [10] protein arrays, or biosensors. [11] In Nanotechnology, amyloids are instrumental in building protein-based materials with enhanced mechanical resistance [12] and conductivity, [13] and their assembly into particles with defined 3D shape might be explored, as it has been implemented with DNA in the origami approach. [14] In [a] Prof. Rafael Giraldo, PhD Department of Chemical & Physical Biology, Program on Biomolecular Recognition & Assembly Centro de Investigaciones Biológicas – CSIC, C/ Ramiro de Maeztu 9, E-28040 Madrid, Spain Fax: (+) 34 91 5360432 E-mail: rgiraldo@cib.csic.es This is the pre-print version of an article finally published as: Giraldo R (2010) Amyloid assemblies: Protein Legos at a crossroads in bottom-up synthetic biology. ChemBioChem 11: 2347-2357. Doi:10.1002/cbic.201000412