Self-assembling nanoscale systems

Self-assembly is ubiquitous in different areas of science, for example in crystals and viruses, and also plays crucial roles in nanotechnology. Many commonalities link these self-assembling systems, in spite of their complexity and different length and time scales. In this thesis, we take an interdisciplinary perspective to gain new insights into self-assembly, exploring ways of modelling self-assembling systems that are relevant across these different fields. A challenge in nanotechnology is to develop self-assembling systems capable of generating a desired outcome. An example is graphene nanoribbons, which are a novel type of semiconductor material with great potential in the nanotech industry. In this context, it is unclear which strategies are best for controlling the output of a self-assembly process, either by manipulation of the thermodynamic environment of the assembling system, or other methods of directing self-assembly. We use quantitative modelling of the kinetics of self-assembly as a tool to predict experimental results in self-assembling systems that are too complex for detailed experimental investigation. Self-assembly of viral protein shells is an example from biology. Viruses have evolved niche methods of assembly that are both robust and highly efficient, as the virus mutation rates are very high, especially in RNA viruses. The viruses discussed in this thesis have an added layer of complexity; it is thought that sequence-specific interactions between viral genomes and the protein building blocks of the viral capsids have a strong impact on the assembly process. We have developed here novel analysis techniques for the modelling of this co-assembly scenario. We use these mechanistic insights to develop new theoretical tools to analyse structural data, providing unprecedented insights into the asymmetric organization of the packaged genome

A group theoretical approach to structural transitions of icosahedral quasicrystals and point arrays

In this paper we describe a group theoretical approach to the study of structural transitions of icosahedral quasicrystals and point arrays. We apply the concept of Schur rotations, originally proposed by Kramer, to the case of aperiodic structures with icosahedral symmetry; these rotations induce a rotation of the physical and orthogonal spaces invariant under the icosahedral group, and hence, via the cut-and-project method, a continuous transformation of the corresponding model sets. We prove that this approach allows for a characterisation of such transitions in a purely group theoretical framework, and provide explicit computations and specific examples. Moreover, we prove that this approach can be used in the case of finite point sets with icosahedral symmetry, which have a wide range of applications in carbon chemistry (fullerenes) and biology (viral capsids).Peer reviewe

Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic beta-oxidation trifunctional enzyme complexes

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid beta-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.Peer reviewe

A novel druggable interprotomer pocket in the capsid of rhino- and enteroviruses

Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are capsid binders that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo-electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens.Peer reviewe

Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug desig

Clustering polymorphs of tau and IAPP fibrils with the CHEP algorithm

Recent steps towards automation have improved the quality and efficiency of the entire cryo-electron microscopy workflow, from sample preparation to image processing. Most of the image processing steps are now quite automated, but there are still a few steps which need the specific intervention of researchers. One such step is the identification and separation of helical protein polymorphs at early stages of image processing. Here, we tested and evaluated our recent clustering approach on three datasets containing amyloid fibrils, demonstrating that the proposed unsupervised clustering method automatically and effectively identifies the polymorphs from cryo-EM images. As an automated polymorph separation method, it has the potential to complement automated helical picking, which typically cannot easily distinguish between polymorphs with subtle differences in morphology, and is therefore a useful tool for the image processing and structure determination of helical proteins

Constraints on the RNA organization consistent with the tomogram.

<p>Each possible RNA organization is characterized by which long edges (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g001" target="_blank">Fig. 1B</a>, purple edges) are occupied in the polyhedral shell of the icosahedrally-averaged density. Long connections are labelled by the numbers of the five-fold vertices (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004146#pcbi.1004146.g001" target="_blank">Fig. 1D</a>) they connect (x•y connecting five-fold vertices x and y). Constraints imposed in the analysis are indicated in the first row, with green indicating an occupied edge, and red an unoccupied edge. The five paths meeting these constraints are characterized according to occupied and non-occupied edges. The last row shows edges shared by all five paths.</p

Classification of polyhedral edges as occupied and non-occupied.

<p>A comparison of the density profiles of the sampled long edge connections. The mean of a fitted normal distribution (y-axis) is scattered with a skewness parameter (x-axis). Connections with negative skew are disregarded as no statement about occupancy can be deduced in this case. From the remainder, two groups of four and five connections are identified as occupied (in the green circle) and non-occupied (red circle), respectively. These are used as constraints in the analysis.</p

The model system—bacteriophage MS2.

<p>(A) The viral capsid is formed from 60 asymmetric and 29 symmetric copies of the CP dimers, with one MP that takes the place of a symmetric dimer (PDBID 2MS2). The genomic RNA is organized inside the particles in two shells, with the outer shell adopting the shape of a polyhedral cage in icosahedrally-averaged reconstructions. (B) Depiction of the polyhedral cage, showing long (purple) and short (orange) PS-PS RNA connections. (C) Asymmetrically averaged tomogram of bacteriophage MS2 bound to its receptor, the bacterial F-pilus. The portion of the electron density corresponding to the CP shell (and bacterial pilus) is shown in blue; green depicts the density for genomic RNA (and presumably some elements of the MP), which forms the basis for the analysis described in this study. The RNA density forms a shell that is intimately associated with the inside surface of the capsid. (D) A planar representation of protein container and polyhedral RNA organization, showing the relative positions of the 60 polyhedral vertices (PS positions, indicated as yellow circles) in contact with the 60 asymmetric CP dimers.</p