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Over half the proteins in the E. coli cytoplasm form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a protein's oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle X-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, some proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins.
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Single particle tracking has found broad applications in the life and physical sciences, enabling the observation and characterization of nano- and microscopic motion. Fluorescence-based approaches are ideally suited for high-background environments, such as tracking lipids or proteins in or on cells, due to superior background rejection. Scattering-based detection is preferable when localization precision and imaging speed are paramount due to the in principle infinite photon budget. Here, we show that micromirror-based total internal reflection dark field microscopy enables background suppression previously only reported for interferometric scattering microscopy, resulting in nanometer localization precision at 6 µs exposure time for 20 nm gold nanoparticles with a 25 × 25 µm2 field of view. We demonstrate the capabilities of our implementation by characterizing sub-nanometer deterministic flows of 20 nm gold nanoparticles at liquid-liquid interfaces. Our results approach the optimal combination of background suppression, localization precision, and temporal resolution achievable with pure scattering-based imaging and tracking of nanoparticles at interfaces.
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The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RING-in-between-RING (RBR) domains, a coordinated ubiquitin relay mechanism between the HOIL-1-interacting protein (HOIP) and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.
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Fatores de Transcrição , Ubiquitina-Proteína Ligases , Ubiquitina , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers-in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.
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Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.
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Microscopia Crioeletrônica/métodos , Espectrometria de Massas/métodos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação ProteicaRESUMO
We study the enhancement of the stiffness of two families of hydrogels (polyacrylamide, PAAm, and polydimethylacrylamide, PDMA) due to the additions of very small amounts of silica nanofillers. It is well established that high concentrations of silica nanoparticles enhance the toughness of both hydrogel types, but significantly more for the PDMA based gels that adsorb readily to silica surfaces. In order to decouple the structural changes in the gels that stem either from polymerization kinetics or from the interactions between nanofillers and polymers, we use a photoinitiator for the polymerization of the composite gels that promotes the structural homogeneity of the hydrogels. We characterize both the mechanical and structural properties of the composite hydrogels as a function of nanofiller concentration, by calculating the single particle diffusion of inert polystyrene tracer particles of three different sizes. In agreement with previous experiments, we find that silica nanoparticles increase the stiffness of PAAm gels more than expected for passive fillers. Surprisingly, we find that a small addition of silica nanoparticles during gel polymerization to PDMA based hydrogels softens them. We attribute this effect to an increase of the average mesh size of the gel, allowing particles of 0.49 µm in diameter to diffuse normally through the gel, but restricting the motion of larger particles. A further increase in silica nanoparticle concentration results in the expected stiffening of the gel. PDMA based composites with a large mean pore size, as reported here, may find applications in particle separation and gentle fixation of microorganisms and cells.
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Actin is a protein that plays an essential role in maintaining the mechanical integrity of cells. In response to strong external stresses, it can assemble into large bundles, but it grows into a fine branched network to induce cell motion. In some cases, the self-organization of actin fibers and networks involves the action of bipolar filaments of the molecular motor myosin. Such self-organization processes mediated by large myosin bipolar filaments have been studied extensively in vitro. Here we create active gels, composed of single actin filaments and small myosin bipolar filaments. The active steady state in these gels persists long enough to enable the characterization of their mechanical properties using one and two point microrheology. We study the effect of myosin concentration on the mechanical properties of this model system for active matter, for two different motor assembly sizes. In contrast to previous studies of networks with large motor assemblies, we find that the fluctuations of tracer particles embedded in the network decrease in amplitude as motor concentration increases. Nonetheless, we show that myosin motors stiffen the actin networks, in accordance with bulk rheology measurements of networks containing larger motor assemblies. This implies that such stiffening is of universal nature and may be relevant to a wider range of cytoskeleton-based structures.
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It is well known that many biochemical processes in the cell such as gene regulation, growth signals and activation of ion channels, rely on mechanical stimuli. However, the mechanism by which mechanical signals propagate through cells is not as well understood. In this review we focus on stress propagation in a minimal model for cell elasticity, actomyosin networks, which are comprised of a sub-family of cytoskeleton proteins. After giving an overview of th actomyosin network components, structure and evolution we review stress propagation in these materials as measured through the correlated motion of tracer beads. We also discuss the possibility to extract structural features of these networks from the same experiments. We show that stress transmission through these networks has two pathways, a quickly dissipative one through the bulk, and a long ranged weakly dissipative one through the pre-stressed actin network.
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We investigate experimentally and theoretically thin layers of colloid particles held adjacent to a solid substrate by gravity. Epifluorescence, confocal, and holographic microscopy, combined with Monte Carlo and hydrodynamic simulations, are applied to infer the height distribution function of particles above the surface, and their diffusion coefficient parallel to it. As the particle area fraction is increased, the height distribution becomes bimodal, indicating the formation of a distinct second layer. In our theory, we treat the suspension as a series of weakly coupled quasi-two-dimensional layers in equilibrium with respect to particle exchange. We experimentally, numerically, and theoretically study the changing occupancies of the layers as the area fraction is increased. The decrease of the particle diffusion coefficient with concentration is found to be weakened by the layering. We demonstrate that particle polydispersity strongly affects the properties of the sedimented layer, because of particle size segregation due to gravity.
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The mechanical properties of polymer gels based on cytoskeleton proteins (e.g. actin) have been studied extensively due to their significant role in biological cell motility and in maintaining the cell's structural integrity. Microrheology is the natural method of choice for such studies due to its economy in sample volume, its wide frequency range, and its spatial sensitivity. In microrheology, the thermal motion of tracer particles embedded in a complex fluid is used to extract the fluid's viscoelastic properties. Comparing the motion of a single particle to the correlated motion of particle pairs, it is possible to extract viscoelastic properties at different length scales. In a recent study, a crossover between intermediate and bulk response of complex fluids was discovered in microrheology measurements of reconstituted actin networks. This crossover length was related to structural and mechanical properties of the networks, such as their mesh size and dynamic correlation length. Here we capitalize on this result giving a detailed description of our analysis scheme, and demonstrating how this relation can be used to extract the dynamic correlation length of a polymer network. We further study the relation between the dynamic correlation length and the structure of the network, by introducing a new length scale, the average filament length, without altering the network's mesh size. Contrary to the prevailing assumption, that the dynamic correlation length is equivalent to the mesh size of the network, we find that the dynamic correlation length increases once the filament length is reduced below the crossover distance.
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Citoesqueleto de Actina/química , Elasticidade , Microfluídica , Movimento (Física) , ViscosidadeRESUMO
In-line holographic optical imaging has the unique capability of high speed imaging in three dimensions at rates limited only by the imaging rate of the camera used. In this technique the 3D data is recorded on the detector in a form of a hologram generated by diffraction between the scattered and unscattered light passing through the sample. For dilute samples of single particles or a small cluster of particles, this technique was shown to result in particle tracking with spatial positioning accuracy of a few nanometers. For dense suspension only approximate reconstruction were achieved with systematic axial positioning errors. We propose a scheme to extend accurate holographic microscopy to dense suspensions, by calibrating the Rayleigh-Sommerfeld reconstruction algorithm against Lorentz-Mie scattering theory. We perform this calibration both numerically and experimentally and define the parameter space in which accurate imaging is achieved, and in which numerical calibration holds. We demonstrate the validity of our approach by imaging two attached particles and measuring the distance between their centers with 36 nm accuracy. A difference of 50 nm in particle diameter is easily measured.
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Coloides/química , Holografia/métodos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia/métodos , Nanopartículas/química , Nanopartículas/ultraestruturaRESUMO
A collection of paromomycin-based di-alkylated cationic amphiphiles differing in the lengths of their aliphatic chain residues were designed, synthesized, and evaluated against 14 Gram positive pathogens that are known to cause skin infections. Paromomycin derivatives that were di-alkylated with C7 and C8 linear aliphatic chains had improved antimicrobial activities relative to the parent aminoglycoside as well as to the clinically used membrane-targeting antibiotic gramicidin D; several novel derivatives were at least 16-fold more potent than the parent aminoglycoside paromomycin. Comparison between a di-alkylated and a mono-alkylated paromomycin indicated that the di-alkylation strategy leads to both an improvement in antimicrobial activity and to a dramatic reduction in undesired red blood cell hemolysis caused by many aminoglycoside-based cationic amphiphiles. Scanning electron microscopy provided evidence for cell surface damage by the reported di-alkylated paromomycins.