Reconstitution of recombinant human CCR4-NOT reveals molecular insights into regulated deadenylation.

CCR4-NOT is a conserved multiprotein complex which regulates eukaryotic gene expression principally via shortening of poly(A) tails of messenger RNA or deadenylation. Here, we reconstitute a complete, recombinant human CCR4-NOT complex. Our reconstitution strategy permits strict compositional control to test mechanistic hypotheses with purified component variants. CCR4-NOT is more active and selective for poly(A) than the isolated exonucleases, CCR4a and CAF1, which have distinct deadenylation profiles in vitro. The exonucleases require at least two out of three conserved non-enzymatic modules (CAF40, NOT10:NOT11 or NOT) for full activity in CCR4-NOT. CAF40 and the NOT10:NOT11 module both bind RNA directly and stimulate deadenylation in a partially redundant manner. Linear motifs from different RNA-binding factors that recruit CCR4-NOT to specific mRNAs via protein-protein interactions with CAF40 can inhibit bulk deadenylation. We reveal an additional layer of regulatory complexity to the human deadenylation machinery, which may prime it either for general or target-specific degradation.

Modeling the Self-Assembly of Protein Complexes through a Rigid-Body Rotational Reaction-Diffusion Algorithm.

The reaction-diffusion equations provide a powerful framework for modeling nonequilibrium, cell-scale dynamics over the long time scales that are inaccessible by traditional molecular modeling approaches. Single-particle reaction-diffusion offers the highest resolution technique for tracking such dynamics, but it has not been applied to the study of protein self-assembly due to its treatment of reactive species as single-point particles. Here, we develop a relatively simple but accurate approach for building rigid structure and rotation into single-particle reaction-diffusion methods, providing a rate-based method for studying protein self-assembly. Our simplifying assumption is that reactive collisions can be evaluated purely on the basis of the separations between the sites, and not their orientations. The challenge of evaluating reaction probabilities can then be performed using well-known equations based on translational diffusion in both 3D and 2D, by employing an effective diffusion constant we derive here. We show how our approach reproduces both the kinetics of association, which is altered by rotational diffusion, and the equilibrium of reversible association, which is not. Importantly, the macroscopic kinetics of association can be predicted on the basis of the microscopic parameters of our structurally resolved model, allowing for critical comparisons with theory and other rate-based simulations. We demonstrate this method for efficient, rate-based simulations of self-assembly of clathrin trimers, highlighting how formation of regular lattices impacts the kinetics of association.

An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes.

Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.

MultiBac: from protein complex structures to synthetic viral nanosystems.

The MultiBac baculovirus/insect cell expression vector system was conceived as a user-friendly, modular tool-kit for producing multiprotein complexes for structural biology applications. MultiBac has allowed the structure and function of many molecular machines to be elucidated, including previously inaccessible high-value drug targets. More recently, MultiBac developments have shifted to customized baculoviral genomes that are tailored for a range of applications, including synthesizing artificial proteins by genetic code expansion. We review some of these developments, including the ongoing rewiring of the MultiBac system for mammalian applications, notably CRISPR/Cas9-mediated gene editing.

His-Tag-Mediated Dimerization of Chemoreceptors Leads to Assembly of Functional Nanoarrays.

Transmembrane chemotaxis receptors are found in bacteria in extended hexagonal arrays stabilized by the membrane and by cytosolic binding partners, the kinase CheA and coupling protein CheW. Models of array architecture and assembly propose receptors cluster into trimers of dimers that associate with one CheA dimer and two CheW monomers to form the minimal "core unit" necessary for signal transduction. Reconstructing in vitro chemoreceptor ternary complexes that are homogeneous and functional and exhibit native architecture remains a challenge. Here we report that His-tag-mediated receptor dimerization with divalent metals is sufficient to drive assembly of nativelike functional arrays of a receptor cytoplasmic fragment. Our results indicate receptor dimerization initiates assembly and precedes formation of ternary complexes with partial kinase activity. Restoration of maximal kinase activity coincides with a shift to larger complexes, suggesting that kinase activity depends on interactions beyond the core unit. We hypothesize that achieving maximal activity requires building core units into hexagons and/or coalescing hexagons into the extended lattice. Overall, the minimally perturbing His-tag-mediated dimerization leads to assembly of chemoreceptor arrays with native architecture and thus serves as a powerful tool for studying the assembly and mechanism of this complex and other multiprotein complexes.

Natural supramolecular protein assemblies.

Supramolecular protein assemblies are an emerging area within the chemical sciences, which combine the topological structures of the field of supramolecular chemistry and the state-of-the-art chemical biology approaches to unravel the formation and function of protein assemblies. Recent chemical and biological studies on natural multimeric protein structures, including fibers, rings, tubes, catenanes, knots, and cages, have shown that the quaternary structures of proteins are a prerequisite for their highly specific biological functions. In this review, we illustrate that a striking structural diversity of protein assemblies is present in nature. Furthermore, we describe structure-function relationship studies for selected classes of protein architectures, and we highlight the techniques that enable the characterisation of supramolecular protein structures.

α-Synuclein aggregation, seeding and inhibition by scyllo-inositol.

Recent literature demonstrates the accelerated aggregation of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease (PD), by the presence of preformed fibrillar conformers in vitro. Furthermore, these preformed fibrillar seeds are suggested to accelerate pathological induction in vivo when injected into the brains of mice. Variation in the results of in vivo studies is proposed to be caused by α-synuclein conformational variants. To investigate the impact of amino acid sequence on seeding efficiency, human and mouse α-synuclein seeds, which vary at 7 amino acid residues, were generated and cross-seeding kinetics studied. Using transmission electron microscopy (TEM), we confirmed that mouse α-synuclein aggregated more rapidly than human α-synuclein. Subsequently, we determined that seeding of human and mouse α-synuclein was more rapid in the presence of seeds generated from the same species. In addition, an established amyloid inhibitor, scyllo-inositol, was examined for potential inhibitory effects on α-synuclein aggregation. TEM analysis of protein:inhibitor assays demonstrated that scyllo-inositol inhibits the aggregation of α-synuclein, suggesting the therapeutic potential of the small molecule in PD.

Evaluation of ß-Amyloid Peptides Fibrillation Induced by Nanomaterials Based on Molecular Dynamics and Surface Plasmon Resonance.

This report investigated the effect of carbon nanomaterials, single-wall carbon nanotube (SWCNT) and graphene oxide, on fibrillation of ß-amyloid 40 (Aß40) based on surface plasmon resonance (SPR) and molecular dynamics (MD). MD simulations are carried out in order to reveal the molecular mechanisms of the interaction between nanomaterials and Aß40. The strong interaction between Aß40 and nanomaterials is related to Van der Waals forces and the Coulomb force, inducing delicate manipulation of the main bonding energy for fibrillation of Aß40. The interaction energy between the Aß peptide and graphene is higher than that of SWCNT. Experimental results show both carbon nanomaterials enhance the appearance of a critical nucleus for nucleation of peptide fibrils. Graphene is more beneficial to assist the nucleation process than SWCNT. Combination of SPR and molecular dynamics could be a high-throughput method to screen protein fibrillation.

Characterization of the direct interaction between KcsA-Kv1.3 and its inhibitors.

Integral membrane proteins (IMPs) are of therapeutic interest and are targeted by a majority of approved drugs. It's difficult to express, purify, and maintain the functional conformation of IMPs. Nanodisc presents a reliable method to solubilize and stabilize IMPs in detergent-free condition. In this study, we demonstrate the assembly and purification of a chimeric ion channel, KcsA-Kv1.3 Nanodisc. We further detail biophysical analysis of the assembled Nanodisc using analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and back scattering interferometry (BSI). AUC is employed to determine the molecular composition of the empty and KcsA-Kv1.3 Nanodisc. Combination of SPR and BSI overcomes each other's limitation and provides insight of equilibrium binding properties of peptide and small molecule ligands to KcsA-Kv1.3.

The lateral diffusion and fibrinogen induced clustering of platelet integrin αIIbß3 reconstituted into physiologically mimetic GUVs.

Platelet integrin αIIbß3 is a key mediator of platelet activation and thrombosis. Upon activation αIIbß3 undergoes significant conformational rearrangement, inducing complex bidirectional signalling and protein recruitment leading to platelet activation. Reconstituted lipid models of the integrin can enhance our understanding of the structural and mechanistic details of αIIbß3 behaviour away from the complexity of the platelet machinery. Here, a novel method of αIIbß3 insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid composition. αIIbß3 was inserted into two GUV lipid compositions that seek to better mimic the platelet membrane. First, "nature's own", comprising 32% DOPC, 25% DOPE, 20% CH, 15% SM and 8% DOPS, intended to mimic the platelet cell membrane. Fluorescence Lifetime Correlation Spectroscopy (FLCS) reveals that exposure of the integrin to the activators Mn(2+) or DTT does not influence the diffusion coefficient of αIIbß3. Similarly, exposure to αIIbß3's primary ligand fibrinogen (Fg) alone does not affect αIIbß3's diffusion coefficient. However, addition of Fg with either activator reduces the integrin diffusion coefficient from 2.52 ± 0.29 to µm(2) s(-1) to 1.56 ± 0.26 (Mn(2+)) or 1.49 ± 0.41 µm(2) s(-1) (DTT) which is consistent with aggregation of activated αIIbß3 induced by fibrinogen binding. The Multichannel Scaler (MCS) trace shows that the integrin-Fg complex diffuses through the confocal volume in clusters. Using the Saffman-Delbrück model as a first approximation, the diffusion coefficient of the complex suggests at least a 20-fold increase in the radius of membrane bound protein, consistent with integrin clustering. Second, αIIbß3 was also reconstituted into a "raft forming" GUV with well defined liquid disordered (Ld) and liquid ordered (Lo) phases. Using confocal microscopy and lipid partitioning dyes, αIIbß3 showed an affinity for the DOPC rich Ld phase of the raft forming GUVs, and was effectively excluded from the cholesterol and sphingomyelin rich Lo phase. Activation and Fg binding of the integrin did not alter the distribution of αIIbß3 between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound αIIbß3 into cholesterol rich domains is not responsible for the integrin clustering observed.

Analysis of selected and designed chimeric D- and L-α-helix assemblies.

D-peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base.

Studying protein assembly with reversible Brownian dynamics of patchy particles.

Assembly of protein complexes like virus shells, the centriole, the nuclear pore complex, or the actin cytoskeleton is strongly determined by their spatial structure. Moreover, it is becoming increasingly clear that the reversible nature of protein assembly is also an essential element for their biological function. Here we introduce a computational approach for the Brownian dynamics of patchy particles with anisotropic assemblies and fully reversible reactions. Different particles stochastically associate and dissociate with microscopic reaction rates depending on their relative spatial positions. The translational and rotational diffusive properties of all protein complexes are evaluated on-the-fly. Because we focus on reversible assembly, we introduce a scheme which ensures detailed balance for patchy particles. We then show how the macroscopic rates follow from the microscopic ones. As an instructive example, we study the assembly of a pentameric ring structure, for which we find excellent agreement between simulation results and a macroscopic kinetic description without any adjustable parameters. This demonstrates that our approach correctly accounts for both the diffusive and reactive processes involved in protein assembly.

Exceptionally stable, redox-active supramolecular protein assemblies with emergent properties.

The designed assembly of proteins into well-defined supramolecular architectures not only tests our understanding of protein-protein interactions, but it also provides an opportunity to tailor materials with new physical and chemical properties. Previously, we described that RIDC3, a designed variant of the monomeric electron transfer protein cytochrome cb562, could self-assemble through Zn(2+) coordination into uniform 1D nanotubes or 2D arrays with crystalline order. Here we show that these 1D and 2D RIDC3 assemblies display very high chemical stabilities owing to their metal-mediated frameworks, maintaining their structural order in ≥90% (vol/vol) of several polar organic solvents including tetrahydrofuran (THF) and isopropanol (iPrOH). In contrast, the unassembled RIDC3 monomers denature in ∼30% THF and 50% iPrOH, indicating that metal-mediated self-assembly also leads to considerable stabilization of the individual building blocks. The 1D and 2D RIDC3 assemblies are highly thermostable as well, remaining intact at up to ∼70 °C and ∼90 °C, respectively. The 1D nanotubes cleanly convert into the 2D arrays on heating above 70 °C, a rare example of a thermal crystalline-to-crystalline conversion in a biomolecular assembly. Finally, we demonstrate that the Zn-directed RIDC3 assemblies can be used to spatiotemporally control the templated growth of small Pt(0) nanocrystals. This emergent function is enabled by and absolutely dependent on both the supramolecular assembly of RIDC3 molecules (to form a periodically organized structural template) and their innate redox activities (to direct Pt(2+) reduction).

Protein-like fully reversible tetramerisation and super-association of an aminocellulose.

Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered "protein-like" and what might be considered as "carbohydrate-like" behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

Interactions of heparin and a covalently-linked antithrombin-heparin complex with components of the fibrinolytic system.

Unfractionated heparin (UFH) is used as an adjunct during thrombolytic therapy. However, its use is associated with limitations, such as the inability to inhibit surface bound coagulation factors. We have developed a covalent conjugate of antithrombin (AT) and heparin (ATH) with superior anticoagulant properties compared with UFH. Advantages of ATH include enhanced inhibition of surface-bound coagulation enzymes and the ability to reduce the overall size and mass of clots in vivo. The interactions of UFH or ATH with the components of the fibrinolytic pathway are not well understood. Our study utilised discontinuous second order rate constant (k2) assays to compare the rates of inhibition of free and fibrin-associated plasmin by AT+UFH vs ATH. Additionally, we evaluated the effects of AT+UFH and ATH on plasmin generation in the presence of fibrin. The k2 values for inhibition of plasmin were 5.74 ± 0.28 x 106 M⁻¹ min⁻¹ and 6.39 ± 0.59 x 106 M⁻¹ min⁻¹ for AT+UFH and ATH, respectively. In the presence of fibrin, the k2 values decreased to 1.45 ± 0.10 x 106 M⁻¹ min⁻¹ and 3.07 ± 0.19 x 106 M⁻¹ min⁻¹ for AT+UFH and ATH, respectively. Therefore, protection of plasmin by fibrin was observed for both inhibitors; however, ATH demonstrated superior inhibition of fibrin-associated plasmin. Rates of plasmin generation were also decreased by both inhibitors, with ATH causing the greatest reduction (approx. 38-fold). Nonetheless, rates of plasmin inhibition were 2-3 orders of magnitude lower than for thrombin, and in a plasma-based clot lysis assay ATH significantly inhibited clot formation but had little impact on clot lysis. Cumulatively, these data may indicate that, relative to coagulant enzymes, the fibrinolytic system is spared from inhibition by both AT+UFH and ATH, limiting reduction in fibrinolytic potential during anticoagulant therapy.

Photocatalytic nanolithography of self-assembled monolayers and proteins.

Self-assembled monolayers of alkylthiolates on gold and alkylsilanes on silicon dioxide have been patterned photocatalytically on sub-100 nm length-scales using both apertured near-field and apertureless methods. Apertured lithography was carried out by means of an argon ion laser (364 nm) coupled to cantilever-type near-field probes with a thin film of titania deposited over the aperture. Apertureless lithography was carried out with a helium-cadmium laser (325 nm) to excite titanium-coated, contact-mode atomic force microscope (AFM) probes. This latter approach is readily implementable on any commercial AFM system. Photodegradation occurred in both cases through the localized photocatalytic degradation of the monolayer. For alkanethiols, degradation of one thiol exposed the bare substrate, enabling refunctionalization of the bare gold by a second, contrasting thiol. For alkylsilanes, degradation of the adsorbate molecule provided a facile means for protein patterning. Lines were written in a protein-resistant film formed by the adsorption of oligo(ethylene glycol)-functionalized trichlorosilanes on glass, leading to the formation of sub-100 nm adhesive, aldehyde-functionalized regions. These were derivatized with aminobutylnitrilotriacetic acid, and complexed with Ni(2+), enabling the binding of histidine-labeled green fluorescent protein, which yielded bright fluorescence from 70-nm-wide lines that could be imaged clearly in a confocal microscope.

"Docking sites": nanometer-scale organization of a reactive, protein-resistant, graft copolymer-based interface for macromolecule immobilization.

The signal-to-noise ratio of a sensor system is determined by the affinity of its active component for the analyte on one hand and its inertness with respect to unrelated stimuli (noise) on the other hand. Nonspecific interactions between the environment and biosensor components (typically constructed from glass, silica, or transition metal oxides) result in nonspecific adsorption onto the latter and constitute a major source of noise. We have previously introduced a polymeric interface for preventing nonspecific adsorption while allowing for high-affinity, specific interactions. It is based on the coassembly of biotinylated and nonbiotinylated poly(l-lysine)-graft-poly(ethylene glycol) from aqueous solutions to negatively charged surfaces, such as Nb(2)O(5). In this study, we investigated by atomic force microscopy the nanoscale organization of this interface for each individual step involved in the preparation of a bioactive interface: polymer adsorption, loading with streptavidin, and binding of biotinylated vesicles.

Study on the interaction between the inclusion complex of hematoxylin with ß-cyclodextrin and DNA.

Ultraviolet-visible (UV-vis) spectra, fluorescence spectra, electrochemistry, and the thermodynamic method were used to discuss the interaction mode between the inclusion complex of hematoxylin with ß-cyclodextrin and herring sperm DNA. On the condition of physiological pH, the result showed that hematoxylin and ß-cyclodextrin formed an inclusion complex with binding ratio n(hematoxylin):n(ß-cyclodextrin) = 1:1. The interaction mode between ß-cyclodextrin-hematoxylin and DNA was a mixed binding, which contained intercalation and electrostatic mode. The binding ratio between ß-cyclodextrin-hematoxylin and DNA was n(ß-cyclodextrin -hematoxylin):n(DNA) = 2:1, binding constant was K(⊖)(298.15K) = 5.29 × 104 L·mol⁻¹, and entropy worked as driven force in this action.

Phased fiber growth in a peptide conjugate: aggregation and disaggregation studies.

A glycine-rich, short pentapeptide conjugate 6, derived from the highly conserved copper-binding octarepeat region of the prion protein, exhibits a tendency to self-aggregate in a time-dependent fashion. Aging of 6 afforded an insight into the phased growth of spherical prefibrillar structures to fibers of long persistence length, as observed by a combination of microscopic techniques. Interestingly, growth of these fibers was inhibited by colchicine, a known inhibitor of microtubule polymerization in a concentration dependent fashion. This study offers an intriguing insight into the occurrence of prefibrillar intermediates on the path to the formation of full length peptide fibers. It is also envisaged that constructs such as 6 may also serve as simple models to study chemical intervention of protein aggregation.

Preparation of nano-sized particles from collagen II by a high-voltage electrostatic field system.

The pilot study describes a novel method for preparing nano-sized particles from collagen II using a high-voltage electrostatic field system. Observations from transmission electron microscopy showed that, in one of the cases, the nano-sized collagen II particles exhibited good sphericity, and the particles were in the range of 23.3+/-1.7 nm in diameter at the experimental setting of 3 kV cm(-1), for a 3 h treatment period and at 25 degrees C (with a collagen concentration of 0.2 mg ml(-1)). When the treatment temperature increased to 30 degrees C, the collagen II began to lose the tendency to form individually separated spherically shaped nano-particles. Moreover, a fibrous structure of collagen II was formed instead of a nano-particle shape at the temperature of 37 degrees C. This result is probably contributed to by an entropy-driven process that is termed fibrillogenesis, a larger force causing the collagen molecules to self-assemble and then form collagen fibrils. It is interesting to note that this is practically the first attempt to produce nano-particles directly from collagen II solution under the treatment of a high-voltage electrostatic field, together with a set of working parameters for the collagen concentration and low-temperature setting.