Journal of Structural Biology

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  • Rotationally invariant image representation for viewing direction classification in cryo-EM
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Zhizhen Zhao , Amit Singer

    We introduce a new rotationally invariant viewing angle classification method for identifying, among a large number of cryo-EM projection images, similar views without prior knowledge of the molecule. Our rotationally invariant features are based on the bispectrum. Each image is denoised and compressed using steerable principal component analysis (PCA) such that rotating an image is equivalent to phase shifting the expansion coefficients. Thus we are able to extend the theory of bispectrum of 1D periodic signals to 2D images. The randomized PCA algorithm is then used to efficiently reduce the dimensionality of the bispectrum coefficients, enabling fast computation of the similarity between any pair of images. The nearest neighbors provide an initial classification of similar viewing angles. In this way, rotational alignment is only performed for images with their nearest neighbors. The initial nearest neighbor classification and alignment are further improved by a new classification method called vector diffusion maps. Our pipeline for viewing angle classification and alignment is experimentally shown to be faster and more accurate than reference-free alignment with rotationally invariant K-means clustering, MSA/MRA 2D classification, and their modern approximations.





    Categories: Journal Articles
  • A marker-free automatic alignment method based on scale-invariant features
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Renmin Han , Fa Zhang , Xiaohua Wan , Jose-Jesus Fernández , Fei Sun , Zhiyong Liu

    In electron tomography, alignment accuracy is critical for high-resolution reconstruction. However, the automatic alignment of a tilt series without fiducial markers remains a challenge. Here, we propose a new alignment method based on Scale-Invariant Feature Transform (SIFT) for marker-free alignment. The method covers the detection and localization of interest points (features), feature matching, feature tracking and optimization of projection parameters. The proposed method implements a highly reliable matching strategy and tracking model to detect a huge number of feature tracks. Furthermore, an incremental bundle adjustment method is devised to tolerate noise data and ensure the accurate estimation of projection parameters. Our method was evaluated with a number of experimental data, and the results exhibit an improved alignment accuracy comparable with current fiducial marker alignment and subsequent higher resolution of tomography.





    Categories: Journal Articles
  • Cryo-EM analysis of the organization of BclA and BxpB in the Bacillus anthracis exosporium
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Cynthia M. Rodenburg , Sylvia A. McPherson , Charles L. Turnbough Jr. , Terje Dokland

    Bacillus anthracis and other pathogenic Bacillus species form spores that are surrounded by an exosporium, a balloon-like layer that acts as the outer permeability barrier of the spore and contributes to spore survival and virulence. The exosporium consists of a hair-like nap and a paracrystalline basal layer. The filaments of the nap are comprised of trimers of the collagen-like glycoprotein BclA, while the basal layer contains approximately 20 different proteins. One of these proteins, BxpB, forms tight complexes with BclA and is required for attachment of essentially all BclA filaments to the basal layer. Another basal layer protein, ExsB, is required for the stable attachment of the exosporium to the spore. To determine the organization of BclA and BxpB within the exosporium, we used cryo-electron microscopy, cryo-sectioning and crystallographic analysis of negatively stained exosporium fragments to compare wildtype spores and mutant spores lacking BclA, BxpB or ExsB (ΔbclA, ΔbxpB and ΔexsB spores, respectively). The trimeric BclA filaments are attached to basal layer surface protrusions that appear to be trimers of BxpB. The protrusions interact with a crystalline layer of hexagonal subunits formed by other basal layer proteins. Although ΔbxpB spores retain the hexagonal subunits, the basal layer is not organized with crystalline order and lacks basal layer protrusions and most BclA filaments, indicating a central role for BxpB in exosporium organization.





    Categories: Journal Articles
  • The structure of the N-terminal domain of the Legionella protein SidC
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Emerich Mihai Gazdag , Stefan Schöbel , Alexander V. Shkumatov , Roger S. Goody , Aymelt Itzen

    The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires’ disease. During infection of eukaryotic cells, the bacterium releases about 300 different bacterial effector molecules that aid in the establishment of the Legionella-containing vacuole (LCV) among which SidC is one of these secreted proteins. However, apart from membrane lipid binding the function of SidC remains elusive. In order to characterize SidC further, we have determined the crystal structure of the N-terminal domain of SidC (amino acids 1–609, referred to as SidC-N) at 2.4Å resolution. SidC-N reveals a novel fold in which 4 potential subdomains (A–D) are arranged in a crescent-like structure. None of these subdomains currently has any known structural homologues, raising the question of how this fold has evolved. These domains are highly interconnected, with a low degree of flexibility towards each other. Due to the extended arrangement of the subdomains, SidC-N may contain multiple binding sites for potential interaction partners.





    Categories: Journal Articles
  • 3D domain swapping in a chimeric c-Src SH3 domain takes place through two hinge loops
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Ana Cámara-Artigas , Sergio Martínez-Rodríguez , Emilia Ortiz-Salmerón , José M. Martín-García

    In the Src Homology 3 domain (SH3) the RT and n-Src loops form a pocket that accounts for the specificity and affinity in binding of proline rich motifs (PRMs), while the distal and diverging turns play a key role in the folding of the protein. We have solved the structure of a chimeric mutant c-Src-SH3 domain where specific residues at the RT- and n-Src-loops have been replaced by those present in the corresponding Abl-SH3 domain. Crystals of the chimeric protein show a single molecule in the asymmetric unit, which appears in an unfolded-like structure that upon generation of the symmetry related molecules reveals the presence of a domain swapped dimer where both, RT- and n-Src loops, act as hinge loops. In contrast, the fold of the diverging type II β-turn and the distal loop are well conserved. Our results are the first evidence for the presence of a structured diverging type II β-turn in an unfolded-like intermediate of the c-Src-SH3 domain, which can be stabilized by interactions from the β-strands of the same polypeptide chain or from a neighboring one. Futhermore, this crystallographic structure opens a unique opportunity to study the effect of the amino acid sequence of the hinge loops on the 3D domain swapping process of c-Src-SH3.





    Categories: Journal Articles
  • Corrigendum to “Structure of MST2 SARAH domain provides insights into its interaction with RAPL” [J. Struct. Biol. 185 (2014) 366–374]
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Guoguang Liu , Zhubing Shi , Shi Jiao , Zhenzhen Zhang , Wenjia Wang , Cuicui Chen , Qian Hao , Meng Zhang , Miao Feng , Liang Xu , Zhen Zhang , Zhaocai Zhou , Min Zhang







    Categories: Journal Articles
  • Correlative Light- and Electron Microscopy with chemical tags
    [Apr 2014]

    Publication date: Available online 31 March 2014
    Source:Journal of Structural Biology

    Author(s): Mario Perkovic , Michael Kunz , Ulrike Endesfelder , Stefanie Bunse , Christoph Wigge , Zhou Yu , Victor-Valentin Hodirnau , Margot P. Scheffer , Anja Seybert , Sebastian Malkusch , Erin M. Schuman , Mike Heilemann , Achilleas S. Frangakis

    Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.





    Categories: Journal Articles
  • Automated particle correspondence and accurate tilt-axis detection in tilted-image pairs
    [Apr 2014]

    Publication date: Available online 30 March 2014
    Source:Journal of Structural Biology

    Author(s): Maxim Shatsky , Pablo Arbelaez , Bong-Gyoon Han , Dieter Typke , Steven E. Brenner , Jitendra Malik , Robert M. Glaeser

    Tilted electron microscope images are routinely collected for an ab initio structure reconstruction as a part of the Random Conical Tilt (RCT) or Orthogonal Tilt Reconstruction (OTR) methods, as well as for various applications using the “free-hand” procedure. These procedures all require identification of particle pairs in two corresponding images as well as accurate estimation of the tilt-axis used to rotate the electron microscope (EM) grid. Here we present a computational approach, PCT (particle correspondence from tilted pairs), based on tilt-invariant context and projection matching that addresses both problems. The method benefits from treating the two problems as a single optimization task. It automatically finds corresponding particle pairs and accurately computes tilt-axis direction even in the cases when EM grid is not perfectly planar.





    Categories: Journal Articles
  • 2dx_automator: Implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline
    [Apr 2014]

    Publication date: Available online 28 March 2014
    Source:Journal of Structural Biology

    Author(s): Sebastian Scherer , Julia Kowal , Mohamed Chami , Venkata Dandey , Marcel Arheit , Philippe Ringler , Henning Stahlberg

    The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein Mlok1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction.





    Categories: Journal Articles
  • A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein
    [Apr 2014]

    Publication date: Available online 28 March 2014
    Source:Journal of Structural Biology

    Author(s): Emma S. Gleave , Helgo Schmidt , Andrew P. Carter

    Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our S accharomyces cerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins.





    Categories: Journal Articles
  • Axial helix rotation as a mechanism for signal regulation inferred from the crystallographic analysis of the E. coli serine chemoreceptor
    [Apr 2014]

    Publication date: Available online 27 March 2014
    Source:Journal of Structural Biology

    Author(s): Hedda U. Ferris , Kornelius Zeth , Michael Hulko , Stanislaw Dunin-Horkawicz , Andrei N. Lupas

    Bacterial chemotaxis receptors are elongated homodimeric coiled-coil bundles, which transduce signals generated in an N-terminal sensor domain across 15–20nm to a conserved C-terminal signaling subdomain. This signal transduction regulates the activity of associated kinases, altering the behavior of the flagellar motor and hence cell motility. Signaling is in turn modulated by selective methylation and demethylation of specific glutamate and glutamine residues in an adaptation subdomain. We have determined the structure of a chimeric protein, consisting of the HAMP domain from Archaeoglobus fulgidus Af1503 and the methyl-accepting domain of Escherichia coli Tsr. It shows a 21nm coiled coil that alternates between two coiled-coil packing modes: canonical knobs-into-holes and complementary x-da, a variant form related to the canonical one by axial rotation of the helices. Comparison of the obtained structure to the Thermotoga maritima chemoreceptor TM1143 reveals that they adopt different axial rotation states in their adaptation subdomains. This conformational change is presumably induced by the upstream HAMP domain and may modulate the affinity of the chemoreceptor to the methylation–demethylation system. The presented findings extend the cogwheel model for signal transmission to chemoreceptors.





    Categories: Journal Articles
  • TRDistiller: A rapid filter for enrichment of sequence datasets with proteins containing tandem repeats
    [Apr 2014]

    Publication date: Available online 26 March 2014
    Source:Journal of Structural Biology

    Author(s): François D. Richard , Andrey V. Kajava

    The dramatic growth of sequencing data evokes an urgent need to improve bioinformatics tools for large-scale proteome analysis. Over the last two decades, the foremost efforts of computer scientists were devoted to proteins with aperiodic sequences having globular 3D structures. However, a large portion of proteins contain periodic sequences representing arrays of repeats that are directly adjacent to each other (so called tandem repeats or TRs). These proteins frequently fold into elongated fibrous structures carrying different fundamental functions. Algorithms specific to the analysis of these regions are urgently required since the conventional approaches developed for globular domains have had limited success when applied to the TR regions. The protein TRs are frequently not perfect, containing a number of mutations, and some of them cannot be easily identified. To detect such “hidden” repeats several algorithms have been developed. However, the most sensitive among them are time-consuming and, therefore, inappropriate for large scale proteome analysis. To speed up the TR detection we developed a rapid filter that is based on the comparison of composition and order of short strings in the adjacent sequence motifs. Tests show that our filter discards up to 22.5% of proteins which are known to be without TRs while keeping almost all (99.2%) TR-containing sequences. Thus, we are able to decrease the size of the initial sequence dataset enriching it with TR-containing proteins which allows a faster subsequent TR detection by other programs. The program is available upon request.





    Categories: Journal Articles
  • Crystallographic snapshot of the Escherichia coli EnvZ histidine kinase in an active conformation
    [Apr 2014]

    Publication date: Available online 26 March 2014
    Source:Journal of Structural Biology

    Author(s): Hedda U. Ferris , Murray Coles , Andrei N. Lupas , Marcus D. Hartmann

    Sensor histidine kinases are important sensors of the extracellular environment and relay signals via conformational changes that trigger autophosphorylation of the kinase and subsequent phosphorylation of a response regulator. The exact mechanism and the regulation of this protein family are a matter of ongoing investigation. Here we present a crystal structure of a functional chimeric protein encompassing the entire catalytic part of the Escherichia coli EnvZ histidine kinase, fused to the HAMP domain of the Archaeoglobus fulgidus Af1503 receptor. The construct is thus equivalent to the full cytosolic part of EnvZ. The structure shows a putatively active conformation of the catalytic domain and gives insight into how this conformation could be brought about in response to sensory input. Our analysis suggests a sequential flip-flop autokinase mechanism.





    Categories: Journal Articles
  • Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins
    [Apr 2014]

    Publication date: Available online 20 March 2014
    Source:Journal of Structural Biology

    Author(s): Martin Humenik , Michael Magdeburg , Thomas Scheibel

    Assembly of recombinant spider silk variants eADF4(Cn) comprising different numbers (n) of the consensus sequence motif C, derived from the natural Araneus diadematus dragline silk ADF4, yielded indistinguishable nanofibrils in cases of n ⩾2. The C-module comprises 35 amino acids rich in glycine and proline residues (in GPGXY repeats) and one polyalanine stretch (Ala)8. All variants were found to be intrinsically disordered in solution, and upon fibril formation they converted into a cross-β structure. Heterologous seeding indicated high structural compatibility between the different eADF4(Cn) variants, however, their assembly kinetics differed in dependence of the number of repeats. Kinetic analysis revealed a nucleation-growth mechanism typical for the formation of cross-β-fibrils, with nucleation rates as well as growth rates increasing with increasing numbers of repeats. Strikingly, the single C-module did not self-assemble into fibrils, but upon addition of heterologous seeds fibril growth could be observed. Apparently, interconnecting of at least two C-modules significantly facilitates the structural transformation from a disordered state into β-sheet structures, which is necessary for nucleation and beneficial for fibril growth.





    Categories: Journal Articles
  • Frealix: Model-based refinement of helical filament structures from electron micrographs
    [Apr 2014]

    Publication date: Available online 20 March 2014
    Source:Journal of Structural Biology

    Author(s): Alexis Rohou , Nikolaus Grigorieff

    The structures of many helical protein filaments can be derived from electron micrographs of their suspensions in thin films of vitrified aqueous solutions. The most successful and generally-applicable approach treats short segments of these filaments as independent “single particles”, yielding near-atomic resolution for rigid and well-ordered filaments. The single-particle approach can also accommodate filament deformations, yielding sub-nanometer resolution for more flexible filaments. However, in the case of thin and flexible filaments, such as some amyloid-β (Aβ) fibrils, the single-particle approach may fail because helical segments can be curved or otherwise distorted and their alignment can be inaccurate due to low contrast in the micrographs. We developed new software called Frealix that allows the use of arbitrarily short filament segments during alignment to approximate even high curvatures. All segments in a filament are aligned simultaneously with constraints that ensure that they connect to each other in space to form a continuous helical structure. In this paper, we describe the algorithm and benchmark it against datasets of Aβ(1–40) fibrils and tobacco mosaic virus (TMV), both analyzed in earlier work. In the case of TMV, our algorithm achieves similar results to single-particle analysis. In the case of Aβ(1–40) fibrils, we match the previously-obtained resolution but we are also able to obtain reliable alignments and ∼8-Å reconstructions from curved filaments. Our algorithm also offers a detailed characterization of filament deformations in three dimensions and enables a critical evaluation of the worm-like chain model for biological filaments.





    Categories: Journal Articles
  • The spectrin family of proteins: A unique coiled-coil fold for various molecular surface properties
    [Apr 2014]

    Publication date: Available online 19 March 2014
    Source:Journal of Structural Biology

    Author(s): Aurélie Nicolas , Olivier Delalande , Jean-François Hubert , Elisabeth Le Rumeur

    The spectrin superfamily is composed of proteins involved in cytolinker functions. Their main structural feature is a large central subdomain with numerous repeats folded in triple helical coiled-coils. Their similarity of sequence was considered to be low without detailed quantification of the intra- and intermolecular levels. Among the superfamily, we considered as essential to propose an overview of the surface properties of all the repeats of the five proteins of the spectrin family, namely α- and β-spectrins, α-actinin, dystrophin and utrophin. Therefore, the aim of this work was to obtain a quantitative comparison of all the repeats at both the primary sequence and the three-dimensional levels. For that purpose, we applied homology modelling methods to obtain structural models for successive and overlapping tandem repeats of the human erythrocyte α- and β-spectrins and utrophin, as previously undertaken for dystrophin, and we used the known structure of α-actinin. The matrix calculation of the pairwise similarities of all the repeat sequences and the electrostatic and hydrophobic surface properties throughout the protein family support the view that spectrins and α-actinin on one hand and utrophin and dystrophin on the other hand share some structural similarities, but a detailed molecular characterisation highlights substantial differences. The repeats within the family are far from identical, which is consistent with their multiple interactions with different cellular partners, including proteins and membrane lipids.





    Categories: Journal Articles
  • Three-dimensional structure of minipig fibrolamellar bone: Adaptation to axial loading
    [Apr 2014]

    Publication date: Available online 14 March 2014
    Source:Journal of Structural Biology

    Author(s): Rotem Almany Magal , Natalie Reznikov , Ron Shahar , Steve Weiner

    Fibrolamellar bone is transiently produced by large, fast growing mammals. The fibrolamellar bone unit is initially formed by elaboration of a network of blood vessels. This is followed by the deposition of a thin, porous and hypercalcified layer, then by the infilling of the vascular cavities by the sequential deposition of a relatively thick rapidly forming bone on both sides of the hypercalcified layer, and finally by lamellar bone. We investigated the 3D structure of the collagenous network of fibrolamellar bone from the femora of a young minipig using mainly the FIB–SEM dual beam microscope and the Serial Surface View method. This enabled us to identify the fibril orientation, the canalicular network organization and other structural motifs within each element of the fibrolamellar unit. The first formed primary hypercalcified layer (PHL) is composed of fibril arrays and multiple small pores, and appears to have an isotropic structure. The major bone component is deposited on both sides of the PHL, and is composed of collagen fibrils with a preferred orientation, mainly aligned parallel to the bone long axis. This bone component is therefore parallel-fibered bone and not woven bone. We also observed that the collagen fibers are organized into bundles. The lamellar bone has most of its collagen fibrils aligned with the bone long axis. This study therefore shows that the large majority of collagen fibrils in fibrolamellar bone are aligned with the bone long axis. This anisotropic structure therefore appears to be adapted to loading along the bone long axis.





    Categories: Journal Articles
  • Two and three dimensional characterization of Zucchini Yellow Mosaic Virus induced structural alterations in Cucurbita pepo L. plants
    [Apr 2014]

    Publication date: Available online 13 March 2014
    Source:Journal of Structural Biology

    Author(s): Günther Zellnig , Michael Herbert Pöckl , Stefan Möstl , Bernd Zechmann

    Infection of plants by Zucchini Yellow Mosaic Virus (ZYMV) induces severe ultrastructural changes. The aim of this study was to investigate ultrastructural changes during ZYMV-infection in Cucurbita pepo L. plants on the two and three dimensional (2D and 3D) level and to correlate these changes with the spread of ZYMV throughout the plant by transmission electron microscopy (TEM) and image analysis. This study revealed that after inoculation of the cotyledons ZYMV moved into roots [3days post inoculation (dpi)], then moved upwards into the stem and apical meristem (5dpi), then into the first true leaf (7dpi) and could finally be found in all plant parts (9dpi). ZYMV-infected cells contained viral inclusion bodies in the form of cylindrical inclusions (CIs). These CIs occurred in four different forms throughout the cytosol of roots and leaves: scrolls and pinwheels when cut transversely and long tubular structures and bundles of filaments when cut longitudinally. 3D reconstruction of ZYMV-infected cells containing scrolls revealed that they form long tubes throughout the cytosol. The majority has a preferred orientation and an average length and width of 3μm and 120nm, respectively. Image analysis revealed an increased size of cells and vacuoles (107% and 447%, respectively) in younger ZYMV-infected leaves leading to a similar ratio of cytoplasm to vacuole (about 1:1) in older and younger ZYMV-infected leaves which indicates advanced cell growth in younger tissues. The collected data advances the current knowledge about ZYMV-induced ultrastructural changes in Cucurbita pepo.





    Categories: Journal Articles
  • Effect of sequence features on assembly of spider silk block copolymers
    [Apr 2014]

    Publication date: Available online 12 March 2014
    Source:Journal of Structural Biology

    Author(s): Olena S. Tokareva , Shangchao Lin , Matthew M. Jacobsen , Wenwen Huang , Daniel Rizzo , David Li , Marc Simon , Cristian Staii , Peggy Cebe , Joyce Y. Wong , Markus J. Buehler , David L. Kaplan

    Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions.





    Categories: Journal Articles
  • Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system
    [Apr 2014]

    Publication date: Available online 12 March 2014
    Source:Journal of Structural Biology

    Author(s): Tim Kükenshöner , Daniel Wohlwend , Christoph Niemöller , Padmarupa Dondapati , Janina Speck , Adebola V. Adeniran , Anita Nieth , Stefan Gerhardt , Oliver Einsle , Kristian M. Müller , Katja M. Arndt

    The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.





    Categories: Journal Articles