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p38, which has been suggested to be a scaffold protein for the assembly of a macromolecular tRNA synthetase complex, contains a leucine zipper-like motif. To understand the importance of the leucine zipper-like motif of p38 (p38LZ) in macromolecular assembly, the p38LZ solution structure was investigated by circular dichroism and nuclear magnetic resonance spectroscopy. The solution structure of p38LZ showed an amphipathic α-helical structure and characteristics similar to a coiled-coil motif. The protein–protein interaction mediated by p38LZ was examined by an in vitro binding assay. The p43 protein, another non-synthetase component of the complex, could bind to p38LZ via its N-terminal domain, which is also predicted to have a potential coiled-coil motif. Thus, we propose that the p38–p43 complex would be formed by coiled-coil interactions, and the formation of the binary complex would facilitate the macromolecular assembly of aminoacyl-tRNA synthetases.

The principles of self-assembly and self-organization are major tenets of molecular and cellular biology. Governed by these principles, the eukaryotic nucleus is composed of numerous subdomains and compartments, collectively described as nuclear bodies. Emerging evidence reveals that associations within and between various nuclear bodies and genomic loci are dynamic and can change in response to cellular signals. This review will discuss recent progress in our understanding of how nuclear body components come together, what happens when they form, and what benefit these subcellular structures may provide to the tissues or organisms in which they are found.

SummaryThe type III secretion system (T3SS) is a large macromolecular assembly found at the surface of many pathogenic Gram-negative bacteria. Its role is to inject toxic “effector” proteins into the cells of infected organisms. The molecular details of the assembly of this large, multimembrane-spanning complex remain poorly understood. Here, we report structural, biochemical, and functional analyses of PrgK, an inner-membrane component of the prototypical Salmonella typhimurium T3SS. We have obtained the atomic structures of the two ring building globular domains and show that the C-terminal transmembrane helix is not essential for assembly and secretion. We also demonstrate that structural rearrangement of the two PrgK globular domains, driven by an interconnecting linker region, may promote oligomerization into ring structures. Finally, we used electron microscopy-guided symmetry modeling to propose a structural model for the intimately associated PrgH-PrgK ring interaction within the assembled basal body.

Protein reconstitution analysis can be useful for studies on protein evolution, protein folding and macromolecular assembly. AroA is a key enzyme in the pathway toward the synthesis of aromatic amino acids in microorganisms and plants, and is the target of the herbicide glyphosate. Our previous study showed that functional AroA enzyme from Escherichia coli could be reconstituted from two ∼220-amino acid fragments of the protein. In this study, we explored this fragment complementation of AroA. Through a systematic study of fragment complementation, we show that successful fragment complementation can be achieved in vivo, when the split sites are within secondary structure elements as well as at loops between structure elements. In addition, we provide evidence, for the first time, that extra ligand, such as glyphosate, can function as a stabilizer of the reconstituted complexes in vivo. Therefore, our results may provide important implications for protein evolution and complex assemblies.

Transcription is the most fundamental step in gene expression in any living organism. Various environmental cues help in the maturation of core RNA polymerase (RNAP; alpha(2)beta beta'omega) with different sigma-factors, leading to the directed recruitment of RNAP to different promoter DNA sequences. Thus it is essential to determine the sigma-factors that affect the preferential partitioning of core RNAP among various a-actors, and the role of sigma-switching in transcriptional gene regulation. Further, the macromolecular assembly of holo RNAP takes place in an extremely crowded environment within a cell, and thus far the kinetics and thermodynamics of this molecular recognition process have not been well addressed. In this study we used a site-directed bioaffinity immobilization method to evaluate the relative binding affinities of three different Escherichia coli sigma-factors to the same core RNAP with variations in temperature and ionic strength while emulating the crowded cellular milieu. Our data indicate that the interaction of core RNAP-sigma is susceptible to changes in external stimuli such as osmolytic and thermal stress, and the degree of susceptibility varies among different sigma-factors. This allows for a reversible sigma-switching from housekeeping factors to alternate sigma-factors when the organism senses a change in its physiological conditions.

The structure of cubic Cowpea mosaic virus crystals, compressed at 330MPa in a diamond anvil cell, was refined at 2.8Å from data collected using ultrashort-wavelength (0.331Å) synchrotron radiation. With respect to the structure at atmospheric pressure, order is increased with lower Debye Waller factors and a larger number of ordered water molecules. Hydrogen-bond lengths are on average shorter and the cavity volume is strongly reduced. A tentative mechanistic explanation is given for the coexistence of disordered and ordered cubic crystals in crystallization drops and for the disorder-order transition observed in disordered crystals submitted to high pressure. Based on such explanation, it can be concluded that pressure would in general improve, albeit to a variable extent, the order in macromolecular crystals.

Within tendon, between collagen fascicles, cells are organized in linear arrays surrounded by a specialized environment of extracellular matrix (ECM) proteins that are largely unidentified. Our goal was to identify interfascicular, pericellular ECM components and provide additional resolution to the organization of the pericellular matrix. To this end, we employed a combination of enzymatic digestion, mechanical disruption, and differential sedimentation to demonstrate for the first time that it possible to liberate living linear tendon cell arrays from whole tendon. Here, we identify type VI collagen, versican, and fibrillin-2 as components of the immediate pericellular ECM of linearly arrayed tendon cells. Additionally, a unique fibrillin-2-containing macromolecular assembly is described in detail for the first time. This new structure is unlike any previously described fibrillin-containing macromolecular assembly. Having a largely constant diameter, it runs axially along tendon cell arrays and can exceed 1000 μm in length.

Membrane protein biogenesis in bacteria occurs via dedicated molecular systems SecYEG and YidC that function independently and in cooperation. YidC belongs to the universally conserved Oxal/Alb3/YidC family of membrane insertases and is believed to associate with translating ribosomes at the membrane surface. Here, we have examined the architecture of the YidC:ribosome complex formed upon YidC-mediated membrane protein insertion. Fluorescence correlation spectroscopy was employed to investigate the complex assembly under physiological conditions. A slightly acidic environment stimulates binding of detergent-solubilized YidC to ribosomes due to electrostatic interactions, while YidC acquires specificity for translating ribosomes at pH-neutral conditions. The nanodisc reconstitution of the YidC to embed it into a native phospholipid membrane environment strongly enhances the YidC: ribosome complex formation. A single copy of YidC suffices for the binding of translating ribosome both in detergent and at the lipid membrane interface, thus being the minimal functional unit. Data reveal molecular details on the insertase functioning and interactions and suggest a new structural model for the YidC:ribosome complex. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

1. Unwin, P. N. T., and R. Henderson. 1975. Molecular structure determination by electron microscopy of unstained crystalline specimens. J. Mol. Biol. 94:425-440. 2. Vainshtein, B. K. 1978. Electron microscopical analysis of the three-dimensional structure of biological macromolecules. Adv. Opt. Electron Microsc. 7:281-377. 3. Steven, A. C., and M. A. Navia. 1982. Specificity of stain distribution in electron micrographs of protein molecules contrasted with uranyl acetate. J. Microsc. 128:145-155. 4. Erickson, H. P. 1973. The Fourier transform of an electron micrograph-first order and second order theory of image formation. Adv. Opt. Electron Microsc. 5:163-199. 5. Engel, A., and A. Massalski. 1984. 3D reconstruction from electron micrographs: Its potential and practical limitations. Ultramicroscopy. 13:71-84. 6. Dorset, D. L., 1984. Dynamical electron scattering from negatively stained protein microcrystals. Ultramicroscopy. 13:311-324. 7. Klug, A. 1978-1979. Image analysis and reconstruction in the electron microscopy of biological macromolecules. Chem. Scripta. 14:245-256. 8. Dorset, D. L., A. Engel, A. Massalski, and J. P. Rosenbusch. 1984. Three-dimensional structure of a membrane pore. Electron microscopical analysis of Escherichia coli outer membrane matrix porin. Biophys. J. 45:128-129. 9. Grinton, G. R., and J. M. Cowley. 1971. Phase and amplitude contrast in electron micrographs of biological material. Optik. 34:221-233. 10. Baldwin, J., and R. Henderson. 1984. Measurement and evaluation of electron diffraction patterns from two-dimensional crystals. Ultramicroscopy. 14:319-336. 11. Cowley, J. M. 1961. Diffraction intensities from bent crystals. Acta Crystallogr. 14:920-927. 12. Dorset, D. L. 1983. Electron crystallography of alkyl chain lipids; identification of long chain packing. Ultramicroscopy. 12:19-28. 13. Dumont, M. E., J. W. Wiggins, and S. B. Hayward. 1980. Electron diffraction of platinum labeled bacteriorhodopsin. Proc. Electron Microsc. Soc. America, 38th Annual Meeting, San Francisco, CA. Claitor's Press, Baton Rouge. 34-35. 14. Doyle, P. A., and P. S. Turner. 1968. Relativistic Hartree-Fock x-ray and electron scattering factors. Acta Crystallogr. A24:390-397.

A brief review of biological electron microscopy at molecular dimensions is given, with emphasis on recent advances in image processing. The advent of frozen-hydrated specimen preparation techniques has made possible the imaging of macromolecular assemblies, such as membrane transport and respiratory chain complexes, in their native state. Analytical techniques are now available which optimize the extraction of structural information from low dose images of ordered and disordered fields of biological macromolecules.

About 250 molecules of the 42,000 molecular weight gene 8 product catalyze the polymerization of the major phage coat protein into a precursor shell temporarily containing both proteins. The resulting prohead appears to be a shell structure with the P8, or scaffolding protein, on the inside, and the coat protein on the outside. In concert with DNA condensation inside the shell, all 250 scaffolding molecules exit from the prohead, without proteolytic cleavage. These molecules then recycle and catalyze the formation of more proheads from newly synthesized coat protein. Such proteins, which catalyze assembly by temporarily associating with an intermediate stage, may represent a general mechanism of macromolecular assembly.

The introduction of the use of T-even bacteriophages as genetic and biochemical experimental systems by Max Delbruck in the late 1930s has led to the intense study of many aspects of bacteriophage biology. Of these, two related endeavors, the study of the structure and the assembly of the virions, have been very important models in the development of our current understanding of macromolecular assembly processes. Twenty years ago, Edgar, Kellenberger, Epstein, and collaborators nucleated these studies by showing that phage assembly follows defined pathways that can accumulate assembly intermediates when blocked and that the assembly-naive components of phage T4 thus accumulated could join properly in vitro (Epstein et al., 1963; Edgar and Wood, 1966; Wood et al., 1968). Since that time, the structure and assembly of many bacteriophages and other viruses have been studied. The possibility of completely defining the genetic systems, and therefore the proteins involved, has made phage assembly a particularly popular and tractable area in which to study macromolecular assembly. We will not consider it the mission of this chapter to collect the details of this myriad of studies. The reader should consult other chapters in this volume or other reviews for such details (e.g., Casjens and King, 1975; Murialdo and Becker, 1978a; Eiserling, 1979; Wood and King, 1979; King, 1980; DuBow, 1981; Mathews et al., 1983; Hendrix et al., 1983; Casjens, 1985c; Carrascosa, 1986). Instead we will focus on general questions currently under study and attempts to answer them in the various dsDNA phage systems. We will not discuss the problem of DNA packaging in detail and will not cover the lipid-containing dsDNA phages.

Tobacco mosaic virus (TMV), a positive-strand RNA virus, provides a unique perspective on nucleic acid structure, in that it has been for some years the only macromolecular assembly in which the structure of a ribonucleic acid interacting with a protein has been visualized in molecular detail, and one of the very few showing protein-nucleic acid interactions at all. Crystallographers have determined the structures of a number of spherical RNA viruses (for references see Liljas, 1986; Stubbs, 1989); but in those structures so far determined, the nucleic acid is not seen in the electron density maps, because it does not conform to the icosahedral symmetry of the coat proteins. TMV, by contrast, is a helical virus, and its RNA is well ordered, with the same symmetry as the protein. It is the type member of the tobamovirus group, rod-shaped viruses 3000 A long and 180 A in diameter, with a central hole of diameter 40 A. Approximately 2130 identical protein subunits of molecular weight 17500 form a right-handed helix of pitch 23A with 49 subunits in 3 turns. A single strand of RNA follows the basic helix between the protein subunits at a radius of 40 A, with three nucleotides bound to each protein subunit. An overall view of part of the viral rod is shown in Figure 5.1.

The status of research on macromolecular assembly is similar in several respects to that of research on macromolecular synthesis in the late 1950’s. The work of that era can teach us some lessons, but it also has left us with some preconceptions that may be misleading us in our attempts to understand assembly mechanisms.

Polyphasic linkage is a close analog of the allosteric and polysteric linkages shown by many biological macromolecules. Like them, it gives rise to both homotropic and heterotropic effects. It is governed by a group of linkage potentials applicable to each separate phase and also, subject to certain conditions, by a group of lower order applicable to the whole system, globally. A good example of polyphasic linkage is provided by sickle cell hemoglobin which, under suitable conditions and subject to control by oxygen, precipitates out of solution to form what appear to be microtubules. This is but one instance of the way in which macromolecular assembly and the formation of subcellular structures generally can be regulated by various small molecules acting as ligands.

The solution conformation of two series of adenosine analogues has been studied by proton HRNMR. The first series comprises 2′ and 3′ deoxy derivatives of the pentoses. In the second series the 2′3′ hydroxyl group of 9β-D-ribo-,ara-and xylo-furanosyl-adenine have been replaced by amino groups. The furanoside ring is analyzed in the AltonaSundaralingam model. It is shown that mainly sterical interactions determine the conformational preference and that the interaction between the 5′CH2OH and the 3′ substituent has the dominant influence.

A microsomal preparation from chick embryo epiphyseal cartilage has been used to study the assembly of the heteropolysaccharide, chondroitin. Endogenous chondroitin sulfate in the microsomal preparation was found to act as a primer for the addition of relatively short non-sulfated polysaccharide chains to form large molecules with a large sulfated portion and a small non-sulfated portion. A smaller primer was found to act as an acceptor for the polymerization of long non-sulfated chains to form large, predominantly non-sulfated chondroitin. Although the incorporation of sugars was time dependent, there was no detectable accumulation of short-chain molecules at the earliest times examined. The results suggest that the mechanism of polysaccharide formation is one of rapid assembly of most of the heteropolysaccharide chain, with subsequent addition taking place at a much slower rate. This would indicate a tightly organized mechanism of synthesis which may represent a general pattern for synthesis of heteropolysaccharides.

The negative-staining technique is being used increasingly in studies of biological membranes, and it is therefore important to investigate the effects of this preparative procedure on the structures of specimens containing lipids.

Macromolecular assembly into complex morphologies and architectural shapes is an area of fundamental research and technological innovation. In this work, we investigate the self-assembly process of recombinantly produced protein inspired by spider silk (spidroin). To elucidate the first steps of the assembly process, we examined highly concentrated and viscous pendant droplets of this protein in air. We show how the protein self-assembles and crystallizes at the water–air interface into a relatively thick and highly elastic skin. Using time-resolved in situ synchrotron x-ray scattering measurements during the drying process, we showed that the skin evolved to contain a high β-sheet amount over time. We also found that β-sheet formation strongly depended on protein concentration and relative humidity. These had a strong influence not only on the amount, but also on the ordering of these structures during the β-sheet formation process. We also showed how the skin around pendant droplets can serve as a reservoir for attaining liquid–liquid phase separation and coacervation from the dilute protein solution. Essentially, this study shows a new assembly route which could be optimized for the synthesis of new materials from a dilute protein solution and determine the properties of the final products.

Proteins have the ability to assemble in multimeric states to perform their specific biological function. Unfortunately, characterizing experimentally these structures at atomistic resolution is usually difficult. For this reason, in silico methodologies aiming at predicting how multiple protein copies arrange to forma multimeric complex would be desirable. We present Parallel OptimizationWorkbench (POW), a swarm intelligence based optimization framework able to deal, in principle, with any optimization problem. We show that POW can be applied to biologically relevant problems such as prediction of protein assemblies and the parameterization of a Coarse-Grained force field for proteins. By combining POW optimizations, Molecular Dynamics simulations, Poisson-Boltzmann calculations and a variety of experiments, we subsequently study two bacterial nanomachieries: Aeromonas hydrophila's pore-forming toxin aerolysin, and Yersinia enterocolitica injectisome. These structures are challenging both for their size, and for the timescales involved in their functioning. Aerolysin is a pore-forming toxin secreted as an hydrophilic monomer. By means of large conformational changes, the protein heptamerizes on the target cell's surface, and finally inserts β-barrel into its lipid bilayer, causing cell death. The main hurdle in the study of this structure is the complexity of the mode of action, which spans timescales currently unreachable by classical molecular dynamics. We show that aerolysin C-terminal region has the dual role of preventing premature oligomerization and helping the folding of tertiary structure, qualifying therefore as an intramolecular chaperone. We study the transmembrane β-barrel properties and compare them with those of the homologous protein α-hemolysin. We show that aerolysin's barrel is more rigid than α-hemolysin's, and should be anion selective. We present models for aerolysin heptamer both in prepore and, for the first time, in membrane-inserted conformation. Our results are validated experimentally, and are consistent with known biochemical and structural data. The injectisome is an example of a type III secretion system. Its most striking feature is probably its size: hundreds of proteins assemble in a unique structure spanning the Gram-negative bacterial double membrane, and protruding outside the cell as a needle for tenth of nanometers. Obtaining an atomistic representation of this massive structure, and therefore some insights about its mode of action, is one of the greatest challenges. We show that the final length of injectisome's needle is determined by the secondary structure content of a ruler protein located inside its cavity during assembly. Using POW, we also produce the first model for Yersinia injectisome's basal body, highlighting the flexibility of this region in adapting between the inner and outer membranes. As a whole, this work demonstrates that a synergy of dry and wet experiments can provide precious insights into macromolecular structure and function.

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of ‘client’ proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease.

Among the different mechanisms used by bacteria to resist antibiotics, active efflux plays a major role. In gram-negative bacteria, active efflux is carried out by tripartite efflux pumps that form a macromolecular assembly spanning both membranes of the cellular wall. At the outer membrane level, a well-conserved Outer Membrane Factor (OMF) protein acts as an exit duct, but its sequence varies greatly among different species. The OMFs share a similar tri-dimensional structure that includes a beta-barrel pore domain that stabilizes the channel within the membrane. In addition, OMFs are often subjected to different N-terminal post-translational modifications, such as an acylation with a lipid. The role of additional N-terminal anchors is all the more intriguing since it is not always required among the OMFs family. Understanding this optional post-translational modification could open new research lines in the field of antibiotics resistance. In E. coli, it has been shown that CusC is modified with a tri-acylated lipid, whereas TolC does not show any. In the case of OprM from Pseudomonas aeruginosa, the N-terminal modification remains a matter of debate, therefore, we used several approaches to investigate this issue. As definitive evidence, we present a new X ray structure at 3.8A resolution that was solved in a new space group, making it possible to model the N-terminal residue as a palmitylated cysteine.

Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP-responsive inner membrane synthase (BcsA), an accessory membrane-anchored protein (BcsB), and several additional Bcs components. Although the BcsAB catalytic duo has been studied in great detail, its interplay with co-expressed subunits remains enigmatic. Here we show that E. coli Bcs proteins partake in a complex protein interaction network. Electron microscopy reveals a stable, megadalton-sized macromolecular assembly, which encompasses most of the inner membrane and cytosolic Bcs components and features a previously unobserved asymmetric architecture. Heterologous reconstitution and mutational analyses point toward a structure–function model, where accessory proteins regulate secretion by affecting both the assembly and stability of the system. Altogether, these results lay the foundation for more comprehensive models of synthase-dependent exopolysaccharide secretion in biofilms and add a sophisticated secretory nanomachine to the diverse bacterial arsenal for virulence and adaptation.

While the activity of multiprotein complexes is crucial for cellular metabolism, little is known about the mechanisms that collectively control the expression of their components. Here, we investigate the regulations targeting the biogenesis of the nuclear pore complex (NPC), the macromolecular assembly mediating nucleocytoplasmic exchanges. Systematic analysis of RNA-binding proteins interactomes, together with in vivo and in vitro assays, reveal that a subset of NPC mRNAs are specifically bound by Hek2, a yeast hnRNP K-like protein. Hek2-dependent translational repression and protein turnover are further shown to finely tune the levels of NPC subunits. Strikingly, mutations or physiological perturbations altering pore integrity decrease the levels of the NPC-associated SUMO protease Ulp1, and trigger the accumulation of sumoylated versions of Hek2 unable to bind NPC mRNAs. Our results support the existence of a quality control mechanism involving Ulp1 as a sensor of NPC integrity and Hek2 as a repressor of NPC biogenesis.

Evolutionary algorithms are heuristic methods that operate on a population of individuals, which represent candidate solutions to optimization problems. Individuals with better quality, or fitness, are reproduced at a higher rate than less fit individuals by recombination and mutation. This process of selection and reproduction, based on fitness, models competition between individuals and leads to survival-of-the-fittest. Although this process lays at the foundation of evolutionary algorithms, the simplistic assumption that a single criterion can describe the quality of individuals is often unmet in real world problems, where multiple objectives or constraints have to be combined in the same fitness function. Furthermore, in nature, survival-of-the-fittest does not derive uniquely from competition, but is better seen as the outcome of an ongoing process of competition in parallel to a process of elimination of non-fit individuals. To resolve this mismatch and model different criteria for individuals separately, the Viability Evolution paradigm has been proposed in the literature. While the idea was previously introduced at a conceptual level, it had not yet been put to the test and lacks experimental validation. Here, we bridged this gap and tested whether viability-inspired evolutionary algorithms could confer advantages over the traditional evolutionary computation paradigm based on fitness. We started by investigating the evolutionary dynamics of a simple viability evolutionary method, and we analyzed the diversity of evolving populations. We also showed that the additional information available to a viability-based algorithm, such as the number of non-viable individuals for each viability criteria, can be exploited during the search for achieving better performance on optimization problems. In particular, we devised viability algorithms for constrained optimization, which outperformed current state-of-the-art methods on a well-known set of constrained optimization benchmarks. Furthermore, we embedded one of our algorithms into a novel framework for predicting structures of protein assemblies at atomic resolution. Finally, we used evolutionary methods to optimize neural networks in the context of a neuroscience investigation on the role of noise in animal behaviors. Noise is a ubiquitous feature of neural systems, but how it affects behaviors is far from being fully understood. After collecting large-scale measurements of Drosophila walking, we used evolutionary algorithms to search for neural networks that could match observed patterns of Drosophila spontaneous and odor-induced walking. By combining dynamical systems analysis and simulations, we showed how a noise-mediated memory of odor exposure accounts for the observed responses. In conclusion, this thesis contributes to evolutionary computation by testing the Viability Evolution paradigm and by proposing efficient viability evolutionary algorithms for constrained optimization. Furthermore, this thesis contributes to biology, by providing a new viability-based framework for predicting protein assemblies and by elucidating a mechanism through which neural noise may shape animal behaviors.

Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation.