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Cryo-Transmission Electron Microscopy Structure of a Gigadalton Peptide Fiber of De Novo Design

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Cryo-Transmission Electron Microscopy Structure of a Gigadalton Peptide Fiber of De Novo Design Cryo-transmission electron microscopy structure of a gigadalton peptide fiber of de novo design Thomas H. Sharpa,b, Marc Bruninga,1, Judith Mantellb,c,1, Richard B. Sessionsb,1, Andrew R. Thomsona,1, Nathan R. Zaccaib,1, R. Leo Bradyb, Paul Verkadeb,c,d,2, and Derek N. Woolfsona,b,2 aSchool of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, United Kingdom; bSchool of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom; cWolfson Bioimaging Facility, University of Bristol, Bristol BS8 1TD, United Kingdom; and dSchool of Physiology and Pharmacology, University of Bristol, Bristol BS8 1TD, United Kingdom Edited by David Baker, University of Washington, Seattle, WA, and approved July 6, 2012 (received for review November 21, 2011) Nature presents various protein fibers that bridge the nanometer of α-helical coiled coils, remain relatively poorly described at to micrometer regimes. These structures provide inspiration for the the assembly, molecular, and atomic levels (7–9). de novo design of biomimetic assemblies, both to address difficul- From a more-applied perspective, interest in peptide- and pro- ties in studying and understanding natural systems, and to provide tein-based fibrous biomaterials has increased recently because routes to new biomaterials with potential applications in nano- these materials have potential applications in biotechnology and technology and medicine. We have designed a self-assembling fi- synthetic biology—for example, as scaffolds for 3D cell culture, ber system, the SAFs, in which two small α-helical peptides are tissue engineering, and templating the assembly of functional in- programmed to form a dimeric coiled coil and assemble in a con- organic materials (10–13). Although natural proteins can and are trolled manner. The resulting fibers are tens of nm wide and tens of being used in these areas, much simpler or stripped-down systems μm long, and, therefore, comprise millions of peptides to give giga- are preferable because they reduce complexity, and potentially dalton supramolecular structures. Here, we describe the structure allow better understanding and control over the folding and of the SAFs determined to approximately 8 Å resolution using cryo- assembly processes leading to fiber formation. Indeed, the past transmission electron microscopy. Individual micrographs show decade has witnessed a plethora of successful peptide-based de- clear ultrastructure that allowed direct interpretation of the pack- signs of fibrous biomaterials encompassing dipeptides, β-hairpins, BIOPHYSICS AND ing of individual α-helices within the fibers, and the construction of protein fragments, peptide-organic hybrids, and peptide amphi- a 3D electron density map. Furthermore, a model was derived using philes, which are based mainly on β-sheet structures (12, 14). COMPUTATIONAL BIOLOGY the cryotransmission electron microscopy data and side chains However, no design process is complete until the structures of the taken from a 2.3 Å X-ray crystal structure of a peptide building components and their assemblies have been determined to high block incapable of forming fibers. This was validated using single- resolution to afford molecular and, ideally, atomistic descrip- particle analysis techniques, and was stable in prolonged molecu- tions. Although in silico models for several of the above systems lar-dynamics simulation, confirming its structural viability. The level have been presented (11, 15, 16) and structures for amyloid-like of self-assembly and self-organization in the SAFs is unprece- assemblies formed from small peptides are being resolved (17, CHEMISTRY dented for a designed peptide-based material, particularly for a 18), there is currently no high-resolution experimental structure system of considerably reduced complexity compared with natural for a fibrous biomaterial of de novo design. proteins. This structural insight is a unique high-resolution descrip- We have used an alternative protein-folding domain, the α-he- tion of how α-helical fibrils pack into larger protein fibers, and pro- lical coiled coil, to design fibrous biomaterials (19). As depicted vides a basis for the design and engineering of future biomaterials. in Fig. 1A, coiled coils comprise two or more α-helical chains that wrap, or supercoil, around one another to form extended fibrous proteins ∣ protein design ∣ helical reconstruction ∣ rope-like structures. Although many of these remain as discrete X-ray crystallography structures (20), others assemble further into supramolecular structures, as seen for example in intermediate filaments (21, 22). ilamentous proteins are ubiquitous in nature. They are found Well-defined sequence-to-structure relationships for coiled coils Facross all kingdoms of life, where they occur within cells, at- are available, which provide a solid basis for rational protein de- tached to cell surfaces, and extracellularly. They perform a wide sign (23). variety of cellular functions, including giving shape, resilience, We have used the understanding of coiled coils to construct a and stability to cells and tissues; acting as highways for intracel- de novo designed self-assembling fiber system (SAF) (24). In lular transport; providing the machinery underpinning cell move- these so-called SAFs, two complementary 28-residue polypep- ment; and producing glutinous materials for cell attachment. tides combine to form a “sticky ended,” parallel, dimeric coiled- Eukaryotic intracellular fibrous components—which include coil building block of dimensions of approximately 2 by 6 nm actin polymers, intermediate filaments, and microtubules—are (Fig. 1A). These assemble to form stiff fibers of approximately amongst the best-studied examples at the structural level. In these 40 μm in length and, intriguingly, thicken to approximately cases, the monomeric building blocks, typically of nm dimensions and tens of kDa in mass, assemble through spontaneous or Author contributions: T.H.S., R.L.B., P.V. and D.N.W. designed research; T.H.S., J.M., A.R.T., assisted processes to form fibers large enough to span cells tens and N.R.Z. performed research; T.H.S., M.B., and R.B.S. analyzed data; and T.H.S. and of micrometers across. Some of these proteins and assemblies D.N.W. wrote the paper. have yielded to structural definition at high resolution. There The authors declare no conflict of interest. are X-ray crystal or electron-diffraction structures for the globu- This article is a PNAS Direct Submission. lar forms of actin (1) and the tubulins (2), and molecular models Data deposition: The atomic coordinates and structure factors have been depoted in the for their fibrous states based on lower-resolution electron density Protein Data Bank, www.pdb.org (PDB ID code 3RA3; EMDB code 1995). maps derived from cryotransmission electron microscopy (cryo- 1M.B., J.M., R.B.S., A.R.T., and N.R.Z. contributed equally to this work. TEM) images (3, 4). Likewise, there are experimental structures 2To whom correspondence may be addressed. E-mail: [email protected] or for the bacterial flagella hook and filament (5, 6). However, [email protected]. others have proved intractable to detailed structural studies. This article contains supporting information online at www.pnas.org/lookup/suppl/ For example, the intermediate filaments, which are assemblies doi:10.1073/pnas.1118622109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1118622109 PNAS Early Edition ∣ 1of6 Downloaded by guest on October 2, 2021 ABC Fig. 1. Structure and hierarchy of α-helical SAFs. (A) Two heterodimer-directing peptides (SAF-p1, SAF-p2a) assemble to form a sticky-ended unit that associates further to form coiled-coil fibrils, which are hexagonally packed within the SAFs. Each peptide has two differently charged regions: In both cases, the N and C terminal halves are predominantly basic and acidic, respectively, and are colored blue and pink accordingly. (B and C) Fibrils associate laterally to form fibers (B) that display striations (C) after staining with heavy-metal salts and visualization by TEM. Scale bars, 2 μm(B) and 50 nm (C). 80 nm in diameter (Fig. 1B). We have presented several designs tive-stain TEM (Figs. 2A and 1C, respectively). The microscope on this theme (24–26) and explored the detailed mechanism of was calibrated at high magnification using a catalase crystal and a assembly (27). Thus far, we have probed the structure of the fi- sample of graphitized carbon. This allowed the striations in the bers to low resolution (approximately 2 nm) by combined nega- cryo-TEM micrographs to be determined accurately as a 41.8 Å tive-stain TEM and X-ray fiber diffraction (26). repeat; this was confirmed using T4 bacteriophage as an in situ These studies give the following working model for how the standard, which has a striation spacing of 40.6 Å. As we have SAFs are structurally organized (Fig. 1A): The sticky-ended noted before (26), this corresponds almost precisely to the length blocks assemble end-to-end into fibrils. These fibrils are most of each SAF peptide when configured as a coiled coil—i.e., α likely extended parallel, dimeric, -helical coiled coils, albeit in- 41.44 Å (¼28 · 1.48 Å, the rise per residue in an α-helical coiled terrupted every 28 residues along each chain by the end of one coil). Thus, coiled-coil peptides are aligned along the long axis of peptide and the start of the next. The fibrils pack hexagonally into the fibers as per our original design (24) and subsequent struc- bundles in the matured fibers. There is considerable order in both tural analyses of stained and dried fibers (26). dimensions of the fibers (Fig. 1C), which we have suggested is Moreover, order in the other fiber dimension was also visible imparted by periodic interfibril side-chain interactions both lat- in the cryo-TEM images. Amongst these, there were striations erally and longitudinally through the fibers (24–26). Although with a periodicity of 18 Å parallel to the fiber long axis (Fig.
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