Well-defined Three-dimensional Structures Research Articles

Calcareous Foraminiferal Shells as a Template for the Formation of Hierarchal Structures of Inorganic Nanomaterials

A microorganism template approach has been explored for the fabrication of various well-defined three-dimensional (3D) structures. However, most of these templates suffer from small size (few μm), difficulty to remove the template, or low surface area, which affect their potential use in different applications or makes industrial scale-up difficult. Conversely, foraminifer's microorganisms are large (up to 200 mm), consist of CaCO3 (easy to dissolve in mild acid), and have a relatively high surface area (≈5 m2 g-1). Herein, we demonstrate the formation of hierarchical structures of inorganic materials using calcareous foraminiferal shells such as Sorites, Globigerinella siphonifera, Lox-ostomina amygdaleformis, Calcarina baculatus or hispida, and Peneroplis planatus. Several techniques, such as thermal decomposition of single-source precursors of metal oxides or sulfides, reduction of metal salts directly on the surfaces, and redox reactions, were used for coating of different shell materials and several hybrid compositions, which possess nanofeatures. Finally, we examined the role of the prepared 3D structures on the reduction of 4-nitrophenol (4-NP), ethanol electrooxidation, and water purification. A remarkable performance was achieved in each application. The hierarchical structure leads to the reduction of 4-NP within several minutes, a 27 mA cm-2 current density peak was obtained for ethanol electrooxidation, and more than 95% of the organic dye contaminants were successfully removed. These results show that using foraminiferal shells offers a new way for designing complex hierarchical structures with unique properties.

Nucleation of β-rich oligomers and β-barrels in the early aggregation of human islet amyloid polypeptide

The self-assembly of human islet amyloid polypeptide (hIAPP) into β-sheet rich amyloid aggregates is associated with pancreatic β-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of α-helical intermediates before the rapid conversion into β-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more α-helices than β-sheets, consistent with ensemble-based experimental measurements. However, in ~4–6% independent simulations, β-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These β-rich oligomers could adopt β-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these β-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. β-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.

Osmolyte Induced Tumorigenesis and Metastasis: Interactions With Intrinsically Disordered Proteins.

In spite of a great deal of work, the biochemical mechanisms underlying tumorigenesis and metastasis are not yet fully understood. Specifically regarding metastasis many authors consider that malignancy is caused by the accumulation of mutations. However, evidence is gathering to show that tumors are composed of heterogeneous cell populations subjected to selective pressures. In this micro evolutionary scenario, intra- and extra-cellular selective pressures will determine which subpopulations of tumor cells will thrive and be able to dissociate from the tumor as autonomous metastatic cells. We propose here that alteration of conformations of transcription factors confer novel non-canonical functions that may induce oncogenesis and metastasis in a mutation independent manner. We argue that the functional plasticity of transcription factors is due to intrinsically disordered domains (IDRs) of proteins. IDRs prevent spontaneous folding of proteins into well-defined three-dimensional structures. Because most transcription factors contain IDRs, each could potentially interact with many ligands. This high degree of functional pleiotropy would then be ultimately responsible for the metastatic phenotype. The conformations of proteins can be altered by chemical chaperones collectively known as osmolytes. Osmolytes are small organic molecules permeable through biological membranes that can accumulate in cells, increase the thermodynamic stability of proteins, modulate enzyme activity and prevent protein aggregation. Thus, by modifying IDRs, osmolytes could subvert the homeostatic regulatory network of cells. Untargeted metabolomic analysis of oral cancer cells showed that those with the greatest metastatic potential contained several osmolytes that were absent in the non-metastatic cells. We hypothesize that high concentrations of osmolytes might promote conformational alterations of transcription factors that favor metastatic behavior. This hypothesis is eminently testable by investigating whether: (a) the intracellular microenvironment of metastatic cells differs from non-metastatic cells and whether osmolytes are responsible for this change and (b) high intracellular concentrations of osmolytes are sufficient to induce structural modifications in regulatory protein so as to establish novel interactive networks that will constitute the metastatic phenotype. Synthetic cell penetrating peptides mimicking IDRs could act as sensitive probes. By exposing the peptides to the microenvironments of living tumor and metastatic tumor cells one should be able to compare the chemical shifts as revealed by spectra obtained by nuclear magnetic resonance (NMR).

Precision structuring and functionalization of ceramics with ultra-short laser pulses

High-performance ceramics have been firmly established for the manufacturing of tools and components in the modern electronics industry and mechatronics. Various components such as circuit boards, bearings, and sensors benefit from their specific characteristics, such as wear resistance, stiffness, and electrical neutrality. Apart from these advantages, the brittleness and hardness of ceramics turn the mechanical processing into a challenging and difficult task. Against this background, modern laser technologies have already been used to process ceramics for many years, enabling a contactless and wear-free machining. However, regarding high precision applications, for instance, the drilling of micro-holes or the fabrication of well-defined cavities and three-dimensional structures, conventional laser processes reach their limits. Especially due to thermal influences of the laser radiation, brittle edges, stresses, and redeposited layers emerge. Ultrashort pulse lasers enable completely new processing qualities in these fields. The extremely short pulse durations within the pico- and femtosecond range lead to nonlinear absorption mechanisms and an almost athermal material removal. Thereby, dielectric materials can be processed precisely and gently. In the course of a comprehensive process study, the beam–material interactions of ultrashort pulses with ceramics have been investigated. Besides the material properties, the ablation process is influenced by a multitude of laser parameters, such as wavelength, pulse overlap, and fluence. In order to reveal the most important variables, the experiments have been conducted by applying modern statistical methods. Using alumina (Al2O3) as an example, it is shown how different parameter regimes lead to disparate process qualities and efficiencies. The generated models have been used to optimize industrially interesting applications, from the separation of ceramic printed circuit boards to the realization of precise design structures.

Structures and dynamics of β-barrel oligomer intermediates of amyloid-beta16-22 aggregation

Accumulating evidence suggests that soluble oligomers are more toxic than final fibrils of amyloid aggregations. Among the mixture of inter-converting intermediates with continuous distribution of sizes and secondary structures, oligomers in the β-barrel conformation – a common class of protein folds with a closed β-sheet – have been postulated as the toxic species with well-defined three-dimensional structures to perform pathological functions. A common mechanism for amyloid toxicity, therefore, implies that all amyloid peptides should be able to form β-barrel oligomers as the aggregation intermediates. Here, we applied all-atom discrete molecular dynamics (DMD) simulations to evaluate the formation of β-barrel oligomers and characterize their structures and dynamics in the aggregation of a seven-residue amyloid peptide, corresponding to the amyloid core of amyloid-β with a sequence of 16KLVFFAE22 (Aβ16-22). We carried out aggregation simulations with various numbers of peptides to study the size dependence of aggregation dynamics and assembly structures. Consistent with previous computational studies, we observed the formation of β-barrel oligomers in all-atom DMD simulations. Using a network-based approach to automatically identify β-barrel conformations, we systematically characterized β-barrels of various sizes. Our simulations revealed the conformational inter-conversion between β-barrels and double-layer β-sheets due to increased structural strains upon forming a closed β-barrel while maximizing backbone hydrogen bonds. The potential of mean force analysis further characterized the free energy barriers between these two states. The obtained structural and dynamic insights of β-barrel oligomers may help better understand the molecular mechanism of oligomer toxicities and design novel therapeutics targeting the toxic β-barrel oligomers. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy

Collapse Transitions of Proteins and the Interplay Among Backbone, Sidechain, and Solvent Interactions.

Proteins can collapse into compact globules or form expanded, solvent-accessible, coil-like conformations. Additionally, they can fold into well-defined three-dimensional structures or remain partially or entirely disordered. Recent discoveries have shown that the tendency for proteins to collapse or remain expanded is not intrinsically coupled to their ability to fold. These observations suggest that proteins do not have to form compact globules in aqueous solutions. They can be intrinsically disordered, collapsed, or expanded, and even form well-folded, elongated structures. This ability to decouple collapse from folding is determined by the sequence details of proteins. In this review, we highlight insights gleaned from studies over the past decade. Using a polymer physics framework, we explain how the interplay among sidechains, backbone units, and solvent determines the driving forces for collapsed versus expanded states in aqueous solvents.

Plants are not so different

Hi-C experiments in rice reveal that the rice genome is partitioned into well-defined three-dimensional structures similar to so-called topologically associating domains found in metazoans.

Conditional Chaperone-Client Interactions Revealed by Genetically Encoded Photo-cross-linkers.

The cell envelope is an integral and essential component of Gram-negative bacteria. As the front line during host-pathogen interactions, it is directly challenged by host immune responses as well as other harsh extracellular stimuli. The high permeability of the outer-membrane and the lack of ATP energy system render it difficult to maintain important biological activities within the periplasmic space under stress conditions. The HdeA/B chaperone machinery is the only known acid resistant system found in bacterial periplasm, enabling enteric pathogens to survive through the highly acidic human stomach and establish infections in the intestine. These two homologous chaperones belong to a fast growing family of conditionally disordered chaperones that conditionally lose their well-defined three-dimensional structures to exert biological activities. Upon losing ordered structures, these proteins commit promiscuous binding of diverse clients in response to environmental stimulation. For example, HdeA and HdeB are well-folded inactive dimers at neutral pH but become partially unfolded to protect a wide array of acid-denatured proteins upon acid stress. Whether these conditionally disordered chaperones possess client specificities remains unclear. This is in part due to the lack of efficient tools to investigate such versatile and heterogeneous protein-protein interactions under living conditions. Genetically encoded protein photo-cross-linkers have offered a powerful strategy to capture protein-protein interactions, showing great potential in profiling protein interaction networks, mapping binding interfaces, and probing dynamic changes in both physiological and pathological settings. Despite great success, photo-cross-linkers that can simultaneously capture the promiscuous binding partners and directly identify the interaction interfaces remain technically challenging. Furthermore, methods for side-by-side profiling and comparing the condition-dependent client pools from two homologous chaperones are lacking. Herein, we introduce our recent efforts in developing a panel of versatile genetically encoded photo-cross-linkers to study the disorder-mediated chaperone-client interactions in living cells. In particular, we have developed a series of proteomic-based strategies relying on these new photo-cross-linkers to systematically compare the client profiles of HdeA and HdeB, as well as to map their interaction interfaces. These studies revealed the mode-of-action, particularly the client specificity, of these two conditionally disordered chaperones. In the end, some recent elegant work from other groups that applied the genetically encoded photo-cross-linking strategy to illuminate important protein-protein interactions within bacterial cell envelope is also discussed.

Shape-Morphing Materials from Stimuli-Responsive Hydrogel Hybrids

The formation of well-defined and functional three-dimensional (3D) structures by buckling of thin sheets subjected to spatially nonuniform stresses is common in biological morphogenesis and has become a subject of great interest in synthetic systems, as such programmable shape-morphing materials hold promise in areas including drug delivery, biomedical devices, soft robotics, and biomimetic systems. Given their ability to undergo large changes in swelling in response to a wide variety of stimuli, hydrogels have naturally emerged as a key type of material in this field. Of particular interest are hybrid systems containing rigid inclusions that can define both the anisotropy and spatial nonuniformity of swelling as well as nanoparticulate additives that can enhance the responsiveness and functionality of the material. In this Account, we discuss recent progress in approaches to achieve well-defined shape morphing in hydrogel hybrids. First, we provide an overview of materials and methods that facilitate fabrication of such systems and outline the geometry and mechanics behind shape morphing of thin sheets. We then discuss how patterning of stiff inclusions within soft responsive hydrogels can be used to program both bending and swelling, thereby providing access to a wide array of complex 3D forms. The use of discretely patterned stiff regions to provide an effective composite response offers distinct advantages in terms of scalability and ease of fabrication compared with approaches based on smooth gradients within a single layer of responsive material. We discuss a number of recent advances wherein control of the mechanical properties and geometric characteristics of patterned stiff elements enables the formation of 3D shapes, including origami-inspired structures, concatenated helical frameworks, and surfaces with nonzero Gaussian curvature. Next, we outline how the inclusion of functional elements such as nanoparticles can enable unique pathways to programmable and even reprogrammable shape-morphing materials. We focus to a large extent on photothermally reprogrammable systems that include one of a variety of additives that serve to efficiently absorb light and convert it into heat, thereby driving the response of a temperature-sensitive hydrogel. Such systems are advantageous in that patterns of light can be defined with very high spatial and temporal resolution in addition to offering the potential for wavelength-selective addressability of multiple different inclusions. We highlight recent advances in the preparation of light-responsive hybrid systems capable of undergoing reprogrammable bending and buckling into well-defined 3D shapes. In addition, we describe several examples where shape tuning of hybrid systems enables control over the motion of responsive hydrogel-based materials. Finally, we offer our perspective on open challenges and future areas of interest for the field.

Guest-Induced Folding and Self-Assembly of Conformationally Adaptive Macrocycles into Nanosheets and Nanotubes.

A conformationally adaptive macrocycle is presented, namely zorb[4]arene, which exists in multiple conformations in the uncomplexed state. The binding cavity of zorb[4]arene is concealed, either due to a collapsed conformation or by self-inclusion. The zorb[4]arene with long alkyl chains manifests itself with surprisingly low melting point and thus exist as an oil at room temperature. Binding of a guest molecule induces the folding and conformational rigidity of zorb[4]arene and leads to well-defined three-dimensional structures, which can further self-assemble into nanosheets or nanotubes upon solvent evaporation, depending on guest molecules and the conformations they can induce.

Soft Lithography Using Nectar Droplets.

In spite of significant advances in replication technologies, methods to produce well-defined three-dimensional structures are still at its infancy. Such a limitation would be evident if we were to produce a large array of simple and, especially, compound convex lenses, also guaranteeing that their surfaces would be molecularly smooth. Here, we report a novel method to produce such structures by cloning the 3D shape of nectar drops, found widely in nature, using conventional soft lithography.The elementary process involves transfer of a thin patch of the sugar solution coated on a glass slide onto a hydrophobic substrate on which this patch evolves into a microdroplet. Upon the absorption of water vapor, such a microdroplet grows linearly with time, and its final size can be controlled by varying its exposure time to water vapor. At any stage of the evolution of the size of the drop, its shape can be cloned onto a soft elastomer by following the well-known methods of molding and cross-linking the same. A unique new science that emerges in our attempt to understand the transfer of the sugar patch and its evolution to a spherical drop is the elucidation of the mechanics underlying the contact of a deformable sphere against a solid support intervening a thin liquid film. A unique aspect of this work is to demonstrate that higher level structures can also be generated by transferring even smaller nucleation sites on the surface of the primary lenses and then allowing them to grow by absorption of water vapor. What results at the end is either a well-controlled distribution of smooth hemispherical lenses or compound structures that could have potential applications in the fundamental studies of contact mechanics, wettability, and even in optics.

What Do We Learn from the Molecular Self-Assembly Process?

What Do We Learn from the Molecular Self-Assembly Process?

Synthesis of efficient gene delivery systems by grafting pegylated alkylcarboxylate chains to PAMAM dendrimers: Evaluation of transfection efficiency and cytotoxicity in cancerous and mesenchymal stem cells.

The applications of polyamidoamine (PAMAM) dendrimers have attracted much attention in biomedicine specially non-viral gene delivery because of thier unique characteristics including hyperbranching, multivalency, and well-defined uniform globular three-dimensional structures. In the current study, in order to enhance the transfection efficiency and reduce the cytotoxicity of PAMAMs, bromoalkylcarboxylates with different chain length (2-bromoacetic, 6-bromohexanoic, 10-bromodecanoic and 16-bromohexadecanoic acids) were covalently conjugated with 10% and 30% of primary amines of generation 4 and 5 (G4 and G5) of PAMAM dendrimers to increase the hydrophobicity of the carrier. At the next stage, the alkylcarboxylate-PAMAMs were pegylaed to further modify the PAMAM structures for biological applications. Obtained results demonstrated that the prepared PAMAM derivatives had particle size around 140 nm with net-positive surface charge. None of the prepared PAMAM-based non-viral vactors exhibited significant hemolytic activity and also cytotoxicity. Meanwhile decahexanoate-PAMAM G4 [G4(16C-10%)] and decanoate-PAMAM G4 conjugated to polyethylene glycol (PEG) (G4[(10C-30%)(10C-PEG)]) showed highest transfection efficiency in murine neuroblastoma (Neuro-2a) cell line, interestingly only the latter had improved transfection efficiency in mesenchymal stem cells (MSCs). This study proved the potential utility of alkylcarboxylate-grafted PAMAM dendrimers (G4 and G5) with or without PEG modification for efficient gene transfer into cancerous cells as well as MSCs.

The Pediocin PA-1 Accessory Protein Ensures Correct Disulfide Bond Formation in the Antimicrobial Peptide Pediocin PA-1.

Peptides, in contrast to proteins, are generally not large enough to form stable and well-defined three-dimensional structures. However, peptides are still able to form correct disulfide bonds. Using pediocin-like bacteriocins, we have examined how this may be achieved. Some pediocin-like bacteriocins, such as pediocin PA-1 and sakacin P[N24C+44C], have four cysteines. There are three possible ways by which the four cysteines may combine to form two disulfide bonds, and the three variants are expected to be produced in approximately equal amounts if their formation is random. Pediocin PA-1 and sakacin P[N24C+44C] with correct disulfide bonds were the main products when they were secreted by the pediocin PA-1 ABC transporter and accessory protein, but when they were secreted by the corresponding secretion machinery for sakacin A, a pediocin-like bacteriocin with one disulfide bond (two cysteines), peptides with all three possible disulfide bonds were produced in approximately equal amounts. All five cysteines in the pediocin PA-1 ABC transporter and the two cysteines (that form a CxxC motif) in the accessory protein were individually replaced with serines to examine their involvement in disulfide bond formation in pediocin PA-1. The Cys86Ser mutation in the accessory protein caused a 2-fold decrease in the amount of pediocin PA-1 with correct disulfide bonds, while the Cys83Ser mutation nearly abolished the production of pediocin PA-1 and resulted in the production of all three disufide bond variants in equal amounts. The Cys19Ser mutation in the ABC transporter completely abolished secretion of pediocin PA-1, suggesting that Cys19 is in the proteolytic active site and involved in cleaving the prebacteriocin. Replacing the other four cysteines in the ABC transporter with serines caused a slight reduction in the overall amount of secreted pediocin PA-1, but the relative amount with the correct disulfide bonds remained large. These results indicate that the pediocin PA-1 accessory protein has a chaperone-like activity in that it ensures the formation of the correct disulfide bond in pediocin PA-1.

Looking for the key to secrets of RNA under the lamp-post.

Looking for the key to secrets of RNA under the lamp-post.

ChemInform Abstract: Constraining Cyclic Peptides to Mimic Protein Structure Motifs

AbstractReview: [172 refs.

CIDER: Classification of Intrinsically Disordered Ensemble Regions

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins fail to fold into well-defined three-dimensional structures as autonomous units. These sequences play important functional roles in signaling pathways, transcriptional regulation, and RNA metabolism. Recent studies have uncovered the physical principles underlying the relationships between IDP / IDR sequences and the range of conformations they adopt. IDP / IDR sequences can be classified into distinct conformational classes based on their amino acid compositions. These classes reflect the sequence-encoded balance between solvent-mediated intrachain electrostatic repulsions and attractions. Specifically, the number and linear sequence distribution of oppositely charged residues partitions the space of IDP / IDR sequences into globules, chimeras of globules and coils, designable random coils, and semi-flexible rod-like conformations.Here, we present CIDER (Classification of Intrinsically Disordered Ensemble Regions) [http://pappulab.wustl.edu/CIDER]. The CIDER webserver provides a rapid and intuitive computational route for designating the appropriate conformational class to a sequence and calculating a number of key parameters that enable inferences regarding conformational properties of IDPs / IDRs. CIDER is freely available and can be used online via a web server to achieve rapid annotation of sequence-disorder relationships for IDPs / IDRs. We also present a freely available non-web version (localCIDER), which we use to perform a high throughput proteomic level sequence analysis to uncover patterns that govern known phosphorylation sites within IDP sequences. The correlation between known phosphosites and the distribution and fraction of charged residues suggests that phosphorylation is used to modulate the underlying charge patterning to engender an expansion of the disordered sequence. This hints at the use of phosphorylation as a reversible switch to toggle IDPs between distinct conformation al classes.

High thermodynamic stability of parametrically designed helical bundles.

We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.

Intrinsically Disordered Proteins: Where Computation Meets Experiment

Proteins are heteropolymers that play important roles in virtually every biological reaction. While many proteins have well-defined three-dimensional structures that are inextricably coupled to their function, intrinsically disordered proteins (IDPs) do not have a well-defined structure, and it is this lack of structure that facilitates their function. As many IDPs are involved in essential cellular processes, various diseases have been linked to their malfunction, thereby making them important drug targets. In this review we discuss methods for studying IDPs and provide examples of how computational methods can improve our understanding of IDPs. We focus on two intensely studied IDPs that have been implicated in very different pathologic pathways. The first, p53, has been linked to over 50% of human cancers, and the second, Amyloid-β (Aβ), forms neurotoxic aggregates in the brains of patients with Alzheimer’s disease. We use these representative proteins to illustrate some of the challenges associated with studying IDPs and demonstrate how computational tools can be fruitfully applied to arrive at a more comprehensive understanding of these fascinating heteropolymers.

Building with alphahelical coiled coils

Protein Design Understanding how proteins fold into well-defined three-dimensional structures has been a longstanding challenge. Increased understanding has led to increased success at designing proteins that mimic existing protein folds. This raises the possibility of custom design of proteins with structures not seen in nature. Thomson et al. describe the design of channelcontaining α-helical barrels, and Huang et al. designed hyperstable helical bundles. Both groups used rational and computational design to make new protein structures based on α-helical coiled coils but took different routes to reach different target structures. Science , this issue p. [485][1], p. [481][2] [1]: /lookup/doi/10.1126/science.1257452 [2]: /lookup/doi/10.1126/science.1257481