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Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process of GV assembly remains elusive. To begin understanding this process, we have devised a high-throughput in vivo assay to determine the interactions of all 11 proteins in the pNL29 GV operon. Complete or partial deletions of the operon establish interdependent relationships among GV proteins during assembly. We also examine the tolerance of the GV assembly process to protein mutations and the cellular burdens caused by GV proteins. Clusters of GV protein interactions are revealed, proposing plausible protein complexes that are important for GV assembly. We anticipate our findings will set the stage for designing GVs that efficiently assemble in heterologous hosts during biomedical applications. Synopsis: Growing interest in biomedical applications for gas vesicles (GVs) demands an understanding of their structure and assembly, but little is known to date on the roles of individual gas vesicle proteins (Gvps) as assembly factors. This study utilizes modular, parallelized protein-protein interaction screening to elucidate key interactions and interdependencies of Gvps during GV assembly. Out of 66 possible Gvp-to-Gvp interactions, 28 formed a dense protein-protein interaction network. Selective inclusion of other Gvps led to gain or loss of protein-protein interactions, revealing interdependence during GV assembly. Specific subnetworks of Gvps are revealed that may be linked to different stages of GV assembly. A number of Gvps show tolerance for fusion proteins during GV assembly, unveiling a degree of plasticity with implications for GV engineering. Iburg at al develop a high-throughput in vivo screen to construct a comprehensive map of the interactions among all 11 proteins of the pNL29 gas vesicle operon. [ABSTRACT FROM AUTHOR]

Macromolecular complexes are often composed of diverse subunits. The self-assembly of these subunits is inherently nonequilibrium and must avoid kinetic traps to achieve high yield over feasible timescales. We show how the kinetics of self-assembly benefits from diversity in subunits because it generates an expansive parameter space that naturally improves the "expressivity" of self-assembly, much like a deeper neural network. By using automatic differentiation algorithms commonly used in deep learning, we searched the parameter spaces of mass-action kinetic models to identify classes of kinetic protocols that mimic biological solutions for productive self-assembly. Our results reveal how high-yield complexes that easily become kinetically trapped in incomplete intermediates can instead be steered by internal design of rate-constants or external and active control of subunits to efficiently assemble. Internal design of a hierarchy of subunit binding rates generates self-assembly that can robustly avoid kinetic traps for all concentrations and energetics, but it places strict constraints on selection of relative rates. External control via subunit titration is more versatile, avoiding kinetic traps for any system without requiring molecular engineering of binding rates, albeit less efficiently and robustly. We derive theoretical expressions for the timescales of kinetic traps, and we demonstrate our optimization method applies not just for design but inference, extracting intersubunit binding rates from observations of yield-vs.- time for a heterotetramer. Overall, we identify optimal kinetic protocols for self-assembly as a powerful mechanism to achieve efficient and high-yield assembly in synthetic systems whether robustness or ease of "designability" is preferred. [ABSTRACT FROM AUTHOR]

The 26S proteasome is the largest and most complicated protease known, and changes to proteasome assembly or function contribute to numerous human diseases. Assembly of the 26S proteasome from its ~66 individual polypeptide subunits is a highly orchestrated process requiring the concerted actions of both intrinsic elements of proteasome subunits, as well as assistance by extrinsic, dedicated proteasome assembly chaperones. With the advent of near-atomic resolution cryo-electron microscopy, it has become evident that the proteasome is a highly dynamic machine, undergoing numerous conformational changes in response to ligand binding and during the proteolytic cycle. In contrast, an appreciation of the role of conformational dynamics during the biogenesis of the proteasome has only recently begun to emerge. Herein, we review our current knowledge of proteasome assembly, with a particular focus on how conformational dynamics guide particular proteasome biogenesis events. Furthermore, we highlight key emerging questions in this rapidly expanding area. [ABSTRACT FROM AUTHOR]

Increasing attention has been focused on the study of protein–metabolite interactions (PMI), which play a key role in regulating protein functions and directing an orchestra of cellular processes. The investigation of PMIs is complicated by the fact that many such interactions are extremely short-lived, which requires very high resolution in order to detect them. As in the case of protein–protein interactions, protein–metabolite interactions are still not clearly defined. Existing assays for detecting protein–metabolite interactions have an additional limitation in the form of a limited capacity to identify interacting metabolites. Thus, although recent advances in mass spectrometry allow the routine identification and quantification of thousands of proteins and metabolites today, they still need to be improved to provide a complete inventory of biological molecules, as well as all interactions between them. Multiomic studies aimed at deciphering the implementation of genetic information often end with the analysis of changes in metabolic pathways, as they constitute one of the most informative phenotypic layers. In this approach, the quantity and quality of knowledge about PMIs become vital to establishing the full scope of crosstalk between the proteome and the metabolome in a biological object of interest. In this review, we analyze the current state of investigation into the detection and annotation of protein–metabolite interactions, describe the recent progress in developing associated research methods, and attempt to deconstruct the very term "interaction" to advance the field of interactomics further. [ABSTRACT FROM AUTHOR]

The 26S proteasome is the largest and most complicated protease known, and changes to proteasome assembly or function contribute to numerous human diseases. Assembly of the 26S proteasome from its ~66 individual polypeptide subunits is a highly orchestrated process requiring the concerted actions of both intrinsic elements of proteasome subunits, as well as assistance by extrinsic, dedicated proteasome assembly chaperones. With the advent of near-atomic resolution cryo-electron microscopy, it has become evident that the proteasome is a highly dynamic machine, undergoing numerous conformational changes in response to ligand binding and during the proteolytic cycle. In contrast, an appreciation of the role of conformational dynamics during the biogenesis of the proteasome has only recently begun to emerge. Herein, we review our current knowledge of proteasome assembly, with a particular focus on how conformational dynamics guide particular proteasome biogenesis events. Furthermore, we highlight key emerging questions in this rapidly expanding area.

Suberin is a specialized cell wall modifying polymer comprising both phenolic-derived and fatty acid-derived monomers, which is deposited in below-ground dermal tissues (epidermis, endodermis, periderm) and above-ground periderm (i.e., bark). Suberized cells are largely impermeable to water and provide a critical protective layer preventing water loss and pathogen infection. The deposition of suberin is part of the skin maturation process of important tuber crops such as potato and can affect storage longevity. Historically, the term "suberin" has been used to describe a polyester of largely aliphatic monomers (fatty acids, ω-hydroxy fatty acids, α,ω-dioic acids, 1-alkanols), hydroxycinnamic acids, and glycerol. However, exhaustive alkaline hydrolysis, which removes esterified aliphatics and phenolics from suberized tissue, reveals a core poly(phenolic) macromolecule, the depolymerization of which yields phenolics not found in the aliphatic polyester. Time course analysis of suberin deposition, at both the transcriptional and metabolite levels, supports a temporal regulation of suberin deposition, with phenolics being polymerized into a poly(phenolic) domain in advance of the bulk of the poly(aliphatics) that characterize suberized cells. In the present review, we summarize the literature describing suberin monomer biosynthesis and speculate on aspects of suberin assembly. In addition, we highlight recent advances in our understanding of how suberization may be regulated, including at the phytohormone, transcription factor, and protein scaffold levels. [ABSTRACT FROM AUTHOR]

Introduction: Cutin is a biopolyester involved in waterproofing aerial plant organs, including leaves. Cutin quantification and compositional profiling require depolymerisation, namely by methanolysis, but specific protocols are not available. Objectives: Investigate how different methanolysis conditions regarding catalyst concentration effect cutin depolymerisation and monomer release, to better define protocols for cutin content determination and composition profiling. Material and Methods: Cork oak (Quercus suber) dewaxed leaves were reacted with five sodium methoxide (NaOMe) concentrations. Extracts were analysed: glycerol by high‐performance liquid chromatography (HPLC) and long‐chain lipids by gas chromatography–mass spectrometry (GC–MS). Results: Cutin was completely removed by 3% NaOMe (8.4% of dewaxed leaves), while mild 0.1% and 0.01% NaOMe methanolysis only depolymerised 14% of total cutin. Reactivity of cutin ester bonds is not homogeneous and glyceridic ester bonds are more easily cleaved, releasing the existing glycerol already under the mildest conditions (0.53% with 0.01% NaOMe and 0.41% with 3% NaOMe). The composition of cutin extracts varies with depolymerisation extent, with easier release of alkanoic acids and alkanols, respectively, 34.9% and 8.8% of total monomers at 0.1% NaOMe, while ω‐hydroxyacids (49.3% of total monomers) and α,ω‐diacids (9.0% of the monomers) are solubilised under more intensive reactive conditions. Conclusion: Cutin of Quercus suber leaves is confirmed as a glyceridic polyester of ω‐hydroxyacids and alkanoic acids, with minor content of α,ω‐diacids, and including coumarate moieties. The protocol for the determination of cutin content and compositional profiling was established regarding catalyst concentration. The molar composition of cutin suggests a macromolecular assembly based on glycerol linked to lipid oligomeric chains with moderate cross‐linking. Cutin from Quercus suber leaves was depolymerised with different sodium methoxide (NaOMe) concentrations. Cutin was completely removed by 3% NaOMe (8.4% of dewaxed leaves), while incomplete solubilisation occurred with milder methanolysis (14% of cutin with 0.1% NaOMe). Reactivity of cutin bonds differs with glyceridic ester bonds being more easily cleaved, and all glycerol is released under the mildest conditions. Cutin extracts composition varies with depolymerisation extent with easier alkanoic acids and alkanols release, while ω‐hydroxyacids and α,ω‐diacids are solubilised with more intensive conditions. [ABSTRACT FROM AUTHOR]

An electroconductive nanoparticles (NPs) film consisting of sodium poly(γ-glutamate) (γ-PGANa) and 3,4-diaminothiophene dihydrochloride (DATh) was simply prepared through macromolecular self-assembly and electropolymerization methods. Firstly, the γ-PGANa and the DATh underwent a complexation to form DATh/γ-PGANa composites through the electrostatic interaction; the DATh/γ-PGANa composites were then self-assembled into DATh/γ-PGANa NPs through decreasing the pH value in aqueous solution. Next, an electroconductive NPs film was formed on the surface of a gold electrode by casting the DATh/γ-PGANa NPs and subsequently electropolymerizing the DATh. Last, horseradish peroxidase and Nafion were cast onto the NPs film to obtain an enzymatic biosensor for H2O2 detection. The electroconductive DATh/γ-PGANa NPs film endowed the biosensor with high sensitivity. Under the optimal conditions, the prepared biosensor showed a linear range from 1 × 10−11 to 1 × 10−4 mol·L−1 with a detection limit of 3 × 10−12 mol·L−1 for H2O2 detection. The biosensor can retain its detection performance for 4 weeks. The response currents of the biosensor changed little after adding the interferent into the analyte. The standard deviation of the response between different biosensors did not exceed 2.5%. [ABSTRACT FROM AUTHOR]

Dynamic assembly and remodeling of actin is critical for many cellular processes during development and stress adaptation. In filamentous fungi and budding yeast, actin cables align in a polarized manner along the mother-to-daughter cell axis, and are essential for the establishment and maintenance of polarity; moreover, they rapidly remodel in response to environmental cues to achieve an optimal system response. A formin at the tip region within a macromolecular complex, called the polarisome, is responsible for driving actin cable polymerization during polarity establishment. This polarisome undergoes dynamic assembly through spatial and temporally regulated interactions between its components. Understanding this process is important to comprehend the tuneable activities of the formin-centered nucleation core, which are regulated through divergent molecular interactions and assembly modes within the polarisome. In this Review, we focus on how intrinsically disordered regions (IDRs) orchestrate the condensation of the polarisome components and the dynamic assembly of the complex. In addition, we address how these components are dynamically distributed in and out of the assembly zone, thereby regulating polarized growth. We also discuss the potential mechanical feedback mechanisms by which the forceinduced actin polymerization at the tip of the budding yeast regulates the assembly and function of the polarisome. [ABSTRACT FROM AUTHOR]

The fungal cell wall serves as the interface between the cell and the environment. Fungal cell walls are composed largely of polysaccharides, primarily glucans and chitin, though in many fungi stress-resistant cell types elaborate additional cell wall structures. Here, we use solid-state nuclear magnetic resonance spectroscopy to compare the architecture of cell wall fractions isolated from Saccharomyces cerevisiae spores and Cryptococcus neoformans melanized cells. The specialized cell walls of these two divergent fungi are highly similar in composition. Both use chitosan, the deacetylated derivative of chitin, as a scaffold on which a polyaromatic polymer, dityrosine and melanin, respectively, is assembled. Additionally, we demonstrate that a previously identified but uncharacterized component of the S. cerevisiae spore wall is composed of triglycerides, which are also present in the C. neoformans melanized cell wall. Moreover, we identify a tyrosine-derived constituent in the C. neoformans wall that, although it is not dityrosine, is a non-pigment constituent of the cell wall. The similar composition of the walls of these two phylogenetically distant species suggests that triglycerides, polyaromatics, and chitosan are basic building blocks used to assemble highly stress-resistant cell walls and the use of these constituents may be broadly conserved in other fungal species. [ABSTRACT FROM AUTHOR]

Understanding the dynamics of macromolecular assemblies in solution, such as Liquid-Liquid Phase Separation (LLPS), represents technologic and fundamental challenges in many fields. In cell biology, such dynamics are of great interest, because of their involvement in subcellular processes. In our study, we aimed to control the assembly of macromolecules in aqueous semi-permeable vesicles, that we named osmosomes, using microfluidics. We developed a microfluidic chip that allows for producting and trapping Giant Unilamellar Vesicles (GUVs) encapsulating macromolecules. This device also allows for modification of the composition of the inner phase and of the membranes of the trapped GUVs. The vesicles are produced from water-in-oil-in-water (w/o/w) double emulsions in less than 20 min after discarding the oil phase. They are highly monodisperse and their diameter can be modulated between 20 and 110 µm by tuning the flow rates of fluid phases. Their unilamellarity is proofed by two techniques: (1) fluorescence quenching experiments and (2) the insertion of the α-hemolysin membrane protein pore. We demonstrate that the internal pH of osmosomes can be tuned in less than 1 min by controlling solvent exchanges through the α-hemolysin pores. The detailed analysis of the exchange kinetics suggests that the microfluidic chip provides an efficient pore formation due to the physical trapping of vesicles and the constant flow rate. Finally, we show a proof of concept for macromolecular assembly within osmosomes by pH-triggered LLPS of wheat proteins within a few minutes. [ABSTRACT FROM AUTHOR]

The fungal cell wall serves as the interface between the cell and the environment. Fungal cell walls are composed largely of polysaccharides, primarily glucans and chitin, though in many fungi stress-resistant cell types elaborate additional cell wall structures. Here, we use solid-state nuclear magnetic resonance spectroscopy to compare the architecture of cell wall fractions isolated from Saccharomyces cerevisiae spores and Cryptococcus neoformans melanized cells. The specialized cell walls of these two divergent fungi are highly similar in composition. Both use chitosan, the deacetylated derivative of chitin, as a scaffold on which a polyaromatic polymer, dityrosine and melanin, respectively, is assembled. Additionally, we demonstrate that a previously identified but uncharacterized component of the S. cerevisiae spore wall is composed of triglycerides, which are also present in the C. neoformans melanized cell wall. Moreover, we identify a tyrosine-derived constituent in the C. neoformans wall that, although it is not dityrosine, is a non-pigment constituent of the cell wall. The similar composition of the walls of these two phylogenetically distant species suggests that triglycerides, polyaromatics, and chitosan are basic building blocks used to assemble highly stress-resistant cell walls and the use of these constituents may be broadly conserved in other fungal species.

Many important gram-negative bacterial pathogens use highly sophisticated type III protein secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.

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.

Oocytes stockpile nuclear pore complexes (NPCs) in cytoplasmic membrane sheets called annulate lamellae (AL) in preparation for rapid cell cycles during embryogenesis. Recently, Hampoelz et al. reported that AL–NPC assembly depends on the coordinated formation, transport, and interaction of biomolecular condensates containing distinct sets of nucleoporins. [ABSTRACT FROM AUTHOR]

A new kind of designed water-compatible molecularly imprinted conductive nanoparticles was prepared by combining macromolecular self-assembly with molecularly imprinting technique. An amphiphilic random copolymer poly(DMA- co -EHA- co -HEA- co -St) containing imino groups and photocrosslinking units was synthesized through free radical copolymerization, which could self-assemble with template molecules (glucose) in aqueous solution to establish nanoparticles embedded with glucose. Meanwhile, AuCl 4 − , as precursor, was added to generate Au NPs in situ because of reducibility of protonated imino groups, leading to the formation of molecularly imprinted polymeric nanoparticles decorated with Au NPs (Au@MIPNs). By simply adjusting the pH value of the assembly solution, Au@MIPNs with different loading amount of Au NPs could be obtained. Electrodeposition was employed to fabricate a robust integration of MIP film with a transducer, then the film was photo-crosslinked by UV irradiation. After extraction of the template molecules, an MIP sensor modified with Au NPs (Au@MIP sensor) was successfully constructed. The presence of Au NPs greatly improved the conductivity of Au@MIP film and allowed direct electrical connection between the recognition cavities and the transducer, which enhanced the sensitivity of the sensor for templates detection. Effect of Au NPs amount on the performance of the Au@MIP sensor was also evaluated. The obtained Au@MIP sensor demonstrated acceptable selectivity and wider linear ranges (two linear ranges from 10 −10 to 10 −8 mol L −1 and 10 −8 to 10 0 mol L −1 with a detection limit of 3×10 −12 mol L −1 ) for glucose detection than MIP sensor without decoration of Au NPs. [ABSTRACT FROM AUTHOR]

The objective of this contribution was to propose a model that would explain the nanocomplexes formation between Human Recombinant Insulin (I) and a polydisperse Chitosan (CS). Such an objective implied exploring I and CS concentration conditions that allowed the formation of complexes with defined and reproducible submicronic dimensions. I-CS complexes were obtained by mixing I and CS solutions at pH 2 and then increasing the pH up to 6 promoting electrostatic interactions between them. Colloidal stages of I and I-CS nano-complexes formation were characterized by dynamic light scattering (DLS), ζ-potential, solutions flow behavior and absorbance measurements. 1·10 −2 %, w/w, of CS allowed covering completely the surface protein aggregates constituting core–shell nano-structures of 200 nm, with a ζ-potential of 17,5 mV. Solution dynamic viscosity results kept relation with different stages of nano-complexation process. Biological activity of I-CS complexes was studied in 3T3-L1 cultured fibroblast showing a delayed and sustained activity as compared to free insulin. I-CS nano-complexes could be an alternative for developing a new generation of drugs allowing I protection from the hostile conditions of the body and increasing its absorption. These findings have basic and practical impacts as they could be exploited to exert the controlled release of I in therapeutic formulations by using the I-CS nano-complexes. [ABSTRACT FROM AUTHOR]

A film consisting of poly(γ-glutamic acid) modified with 3-aminothiophene (ATh-γ-PGA) was prepared by macromolecular selfassembly and electropolymerization. ATh-γ-PGA is amphiphilic and electrically conductive. The copolymers undergo selfassembly to form nanoparticles (NPs) on decreasing the pH value of an aqueous solution. A conducting film of NPs was formed on the surface of a gold electrode by casting the ATh-γ-PGA NPs and subsequently electropolymerizing the thiophene units. Next, horseradish peroxidase and Nafion were cast onto the film to obtain an enzymatic biosensor for H2O2. Due to the electropolymerization step, a cross-conjugated polymer network is created that improves electron transfer rates and thus enhances the response. This endows the biosensor with high sensitivity. Two linear ranges are present, the first ranging from 1 x 10-11 to 1 x 10-8 mol⋅L-1, and the second from 1 x 10-8 to 1 x 10-5 mol⋅L-1. The detection limit is as low as 3 x 10-12 mol⋅L-1. The sensor is stable, repeatable, and was successfully applied to the determination of H2O2 in a commercial disinfecting solution. [ABSTRACT FROM AUTHOR]

The plasma membrane H+-ATPase was purified from the yeast K. lactis. The oligomeric state of the H+-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H+-ATPase hexamer in both MASs as the sole/main oligomeric state—in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H+-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H+-ATPase. It is concluded that the quaternary structure of the H+-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.

Bacterial ribosome biogenesis has been an active area of research for more than 30 years and has served as a test-bed for the development of new biochemical, biophysical and structural techniques to understand macromolecular assembly generally. Recent work inspecting the process in vivo has advanced our understanding of the role of ribosome biogenesis factors, the co-transcriptional nature of assembly, the kinetics of the process under sub-optimal conditions, and the rRNA folding and ribosome protein binding pathways. Additionally, new structural work enabled by single-particle electron microscopy has helped to connect in vitro ribosomal protein binding maps to the underlying RNA. This review summarizes the state of these in vivo studies, provides a kinetic model for ribosome assembly under sub-optimal conditions, and describes a framework to compare newly emerging assembly intermediate structures. This article is part of the themed issue 'Perspectives on the ribosome'. [ABSTRACT FROM AUTHOR]

The recent discovery of several forms of higher order protein structures in cells has shifted the paradigm of how we think about protein organization and metabolic regulation. These dynamic and controllable protein assemblies, which are composed of dozens or hundreds of copies of an enzyme or related enzymes, have emerged as important players in myriad cellular processes. We are only beginning to appreciate the breadth of function of these types of macromolecular assemblies. These higher order structures, which can be assembled in response to varied cellular stimuli including changing metabolite concentrations or signaling cascades, give the cell the capacity to modulate levels of biomolecules both temporally and spatially. This provides an added level of control with distinct kinetics and unique features that can be harnessed as a subtle, yet powerful regulatory mechanism. Due, in large part, to advances in structural methods, such as crystallography and cryo-electron microscopy, and the advent of super-resolution microscopy techniques, a rapidly increasing number of these higher order structures are being identified and characterized. In this review, we detail what is known about the structure, function and control mechanisms of these mesoscale protein assemblies, with a particular focus on those involved in purine and pyrimidine metabolism. These structures have important implications both for our understanding of fundamental cellular processes and as fertile ground for new targets for drug discovery and development. [ABSTRACT FROM AUTHOR]

An important step towards achieving functional diversity of biomimetic surfaces is to better understand the co-assembly of the extracellular matrix components. For this, we study type-I and type-III collagen, the two major collagen types in the extracellular matrix. By using atomic force microscopy, custom image analysis, and kinetic modeling, we study their homotypic and heterotypic assembly. We find that the growth rate and thickness of heterotypic fibrils decrease as the fraction of type-III collagen increases, but the fibril nucleation rate is maximal at an intermediate fraction of type-III. This is because the more hydrophobic type-I collagen nucleates fast and grows in both longitudinal and lateral directions, whereas more hydrophilic type-III limits lateral growth of fibrils, driving more monomers to nucleate additional fibrils. This demonstrates that subtle differences in physico-chemical properties of similar molecules can be used to fine-tune their assembly behavior. [ABSTRACT FROM AUTHOR]

Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiological states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.

Summary Members of the YidC/Oxa1/Alb3 family universally facilitate membrane protein biogenesis, via mechanisms that have thus far remained unclear. Here, we investigated two crucial functional aspects: the interaction of YidC with ribosome:nascent chain complexes (RNCs) and the structural dynamics of RNC-bound YidC in nanodiscs. We observed that a fully exposed nascent transmembrane domain (TMD) is required for high-affinity YidC:RNC interactions, while weaker binding may already occur at earlier stages of translation. YidC efficiently catalyzed the membrane insertion of nascent TMDs in both fluid and gel phase membranes. Cryo-electron microscopy and fluorescence analysis revealed a conformational change in YidC upon nascent chain insertion: the essential TMDs 2 and 3 of YidC were tilted, while the amphipathic helix EH1 relocated into the hydrophobic core of the membrane. We suggest that EH1 serves as a mechanical lever, facilitating a coordinated movement of YidC TMDs to trigger the release of nascent chains into the membrane. [ABSTRACT FROM AUTHOR]

To model large biomolecular systems, such as cell and organelles an atomistic description is not currently achievable and is not generally practical. Therefore, simplified coarse-grained (CG) modelling becomes a necessity. One of the most important cellular components is chromatin, a large DNA–protein complex where DNA is highly compacted. Recent progress in coarse graining modelling of the major chromatin components, double helical DNA and the nucleosome core particle (NCP) is presented. First, general principles and approaches allowing rigorous bottom-to-top generation of interaction potentials in the CG models are presented. Then, recent CG models of DNA are reviewed and their adequacy is benchmarked against experimental data on the salt dependence of DNA flexibility (persistence length). Furthermore, a few recent CG models of the NCP are described and their application for studying salt-dependent NCP–NCP interaction is discussed. An example of a multiscale approach to CG modelling of chromatin is presented where interactions and self-assembly of thousands of NCPs in solution are observed. [ABSTRACT FROM AUTHOR]