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WIREs Nanomed Nanobiotechnol
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Protein nanomachines assembly modes: cell‐free expression and biochip perspectives

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Large macromolecular assemblies are widespread in all cell types with diverse structural and functional roles. Whether localized to membranes, nuclei, or cytoplasm, multimeric protein–nucleic acid complexes may be viewed as sophisticated nanomachines, an inspiration to chemical design. The formation of large biological assemblies follows a complex and hierarchical self‐assembly process via ordered molecular recognition events. Serving a paradigm for biological assembly, extensive past studies of T4 bacteriophage and bacterial ribosomes by many groups have been revealing distinct design strategies, yet these two very different multimeric complexes share common mechanistic motifs. An emerging biochip approach highlights two conceptual notions to promote the study of assembly pathways: cell‐free expression provides coupling between synthesis and assembly; surface anchoring allows high‐resolution imaging of structural intermediates and opens up opportunities for rewiring a network by defining unnatural scaffolds for synthetic design applications. WIREs Nanomed Nanobiotechnol 2013, 5:613–628. doi: 10.1002/wnan.1227 This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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Cell‐free multi‐protein assembly. (a) Cell‐free synthesis of bacteriophage T7 occurs in a test tube supplied with T7 genome and E. coli extract. Transcription (TX), translation (TL) and DNA replication (Rep) support high yields of infectious bacteriophage particles, imaged by TEM at a 59000x magnification. Inset shows a blow‐up of a single phage particle. (Reprinted with permission from Ref . Copyright 2012 American Chemical Society) (b) Cell‐free transcription of nine genes constituting one operon of the F1Fo‐ATP synthase results in synthesis of a long mRNA. Translation of the nine gene products results in proteins that self‐assemble to a functional enzyme in the presence of detergents or lipids. The structure assembles properly as visualized by TEM and its enzymatic activity is monitored by a spectroscopic assay (graph). (Reprinted with permission from Ref . Copyright 2011 Elsevier, Ltd.)
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TEM imaging of ribosomal particles. (a) The procedure to capture in vitro assembly intermediates of purified ribosomal RNA and proteins is depicted schematically. (b) TEM images of these particle intermediates are arranged in an array. Each row is a collection of a single time point, with particle averages arranged from left to right by their decreasing population. A heat map indicates the percentage of particles that populate a given class; red: 8–11%, yellow: 6–8%, green: 4–6%, blue: 2–4%. (Reprinted with permission from Ref . Copyright 2010 American Association for the Advancement of Science)
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T4 Baseplate assembly. (a) The assembly pathway as deciphered by many in vitro and in vivo studies of T4 baseplate starts with the formation of wedges and a hub. Six wedges join one hub particle, followed by binding of additional proteins to form the complete baseplate. (b) TEM image of structures resulting from in vitro assembly of the seven proteins constituting the wedge. Baseplate‐like structures of six wedges spontaneously form in the absence of additional proteins. (Reprinted with permission from Ref . Copyright 2010 Elsevier Ltd.)
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Self‐assembly modes. A simplified assembly scheme for three subunits, blue, orange and green. Orange binds blue specifically and strongly (a) and green competes weakly by binding to blue (b). In a thermodynamically driven self‐assembly, despite interactions of green and blue, only the correct assembly is formed (d). Alternatively, the same final ternary complex can be obtained in a facilitated self‐assembly mode, for example by adding green to the reaction pool only after assembly of orange and blue had occurred (c).
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Segmental assembly of large multimeric complexes. (a) A schematic depiction of the assembly of 30S (top) and 50S (bottom) E. coli ribosomal subunits. For both complexes, ribosomal proteins bind to an RNA core in a sequential manner, with some proteins binding directly to the RNA, and more proteins joining the complex in two later steps. The two complexes then coalesce to one large structure, usually in the presence of mRNA (not shown for clarity). (b) The assembly of T4 bacteriophage occurs in two major steps, (i) binding of the capsid (brown particle) to a tail assembly and (ii) tail fibers are added to the capsid‐tail particle. Each of the segments is preassembled in a sequential manner. Sequential tail assembly is depicted (left) starting from assembly of wedges (turquoise) around a hub (brown) to form the baseplate, from which tail inner tube and tail sheath (yellow) grow and are then capped (blue).
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DNA and RNA brushes. (a) Proteins expressed from a DNA brush can assemble and bind to their specific traps (Y shapes) but their assembly would be more localized if originates from surface anchored mRNA brush (b). Within a DNA brush (inset in a), transcription levels are sensitive to the orientation of promoters, with IN orientation (arrows pointing down) resulting in higher transcription levels than OUT (arrows pointing up). Differences have been attributed to formation of local RNA polymerase gradients (orange particles) that are dictated by DNA brush density and promoter orientation.
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Cell‐free expression and assembly of T4 tail proteins. Cell‐free expression of T4 tail protein gp18 (a, yellow monomers) and gp15 (b, blue monomers) in a test tube results in their assembly into tubes (a) and hexameric rings (b) respectively. Co‐expression of the two proteins yields novel structures resembling nanodonoughts (c). Cell‐free expression of gp18 monomers in the presence of surface‐patterned antibodies (red square) results in localized assembly of nanotubes at the anchored traps with very low nonspecific surface binding to nonpatterned surfaces. The border between patterned and non‐patterned surfaces is highlighted (orange line) (d). (Reprinted with permission from Ref . Copyright 2012 Rights Managed by Nature Publishing Group, Ref . Copyright 2007 American Chemical Society)
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Schematics of a TEM biochip. (a) The TEM silicon‐based grid is composed of 70 µm × 70µm supporting an 8‐nm‐thin SiO2 membrane. (b) The grid is irradiated with 365 nm ultraviolet light (purple arrows) through a mask, removing protected moieties from a molecular coating on the SiO2 membrane (purple rectangle). (c) Individual protected (grey) and deprotected (purple) molecules are shown at the photolithographic interface. (d) Chemical structure of the molecular coating. Ultraviolet (UV) irradiation cleaves the amide bond (arrow) and exposes amines for subsequent conjugation with biomolecules. (e) Long DNA polymers encoding a protein of interest are attached to the surface at one end and fluorescently labeled on the other end (red spheres). (f) Antibodies specific to the protein of interest (blue Y shapes) are immobilized next to the DNA. Using cell extracts, DNA is transcribed into mRNA (red coil) by T7 RNA polymerase (brown ellipsoid), and translated into nascent polypeptide (green chain) by E. coli ribosome (dual orange ellipsoids). The folded protein (green folded chain) diffuses and binds to the antibody trap. (Reprinted with permission from Ref . Copyright 2012 Rights Managed by Nature Publishing Group)
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