RESUMEN
Biomolecules composed of a limited set of chemical building blocks can colocalize into distinct, spatially segregated compartments known as biomolecular condensates. While many condensates are known to form spontaneously via phase separation, it has been unclear how immiscible condensates with precisely controlled molecular compositions assemble from a small number of chemical building blocks. We address this question by establishing a connection between the specificity of biomolecular interactions and the thermodynamic stability of coexisting condensates. By computing the minimum interaction specificity required to assemble condensates with target molecular compositions, we show how to design heteropolymer mixtures that produce compositionally complex condensates by using only a small number of monomer types. Our results provide insight into how compositional specificity arises in naturally occurring multicomponent condensates and demonstrate a rational algorithm for engineering complex artificial condensates from simple chemical building blocks.
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In many biopolymer solutions, attractive interactions that stabilize finite-sized clusters at low concentrations also promote phase separation at high concentrations. Here we study a model biopolymer system that exhibits the opposite behavior, whereby self-assembly of DNA oligonucleotides into finite-sized, stoichiometric clusters tends to inhibit phase separation. We first use microfluidics-based experiments to map a novel phase transition in which the oligonucleotides condense as the temperature increases at high concentrations of divalent cations. We then show that a theoretical model of competition between self-assembly and phase separation quantitatively predicts changes in experimental phase diagrams arising from DNA sequence perturbations. Our results point to a general mechanism by which self-assembly shapes phase boundaries in complex biopolymer solutions.
Asunto(s)
ADN , Modelos Químicos , Transición de Fase , ADN/química , Calor , Oligonucleótidos/química , Separación de FasesRESUMEN
Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is underexplored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.
Asunto(s)
Proteínas que Contienen Bromodominio , Proteínas de Ciclo Celular , Cromatina , Proteínas Nucleares , Factores de Transcripción , Humanos , Acetilación , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Termodinámica , Factores de Transcripción/metabolismoRESUMEN
Phase-separated biomolecular condensates exhibit a wide range of dynamic properties, which depend on the sequences of the constituent proteins and RNAs. However, it is unclear to what extent condensate dynamics can be tuned without also changing the thermodynamic properties that govern phase separation. Using coarse-grained simulations of intrinsically disordered proteins, we show that the dynamics and thermodynamics of homopolymer condensates are strongly correlated, with increased condensate stability being coincident with low mobilities and high viscosities. We then apply an "active learning" strategy to identify heteropolymer sequences that break this correlation. This data-driven approach and accompanying analysis reveal how heterogeneous amino acid compositions and nonuniform sequence patterning map to a range of independently tunable dynamic and thermodynamic properties of biomolecular condensates. Our results highlight key molecular determinants governing the physical properties of biomolecular condensates and establish design rules for the development of stimuli-responsive biomaterials.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Aprendizaje Basado en Problemas , Termodinámica , Aminoácidos , Materiales BiocompatiblesRESUMEN
Chemically driven fluids can demix to form condensed droplets that exhibit phase behaviors not observed at equilibrium. In particular, nonequilibrium interfacial properties can emerge when the chemical reactions are driven differentially between the interior and exterior of the phase-separated droplets. Here, we use a minimal model to study changes in the interfacial tension between coexisting phases away from equilibrium. Simulations of both droplet nucleation and interface roughness indicate that the nonequilibrium interfacial tension can either be increased or decreased relative to its equilibrium value, depending on whether the driven chemical reactions are accelerated or decelerated within the droplets. Finally, we show that these observations can be understood using a predictive theory based on an effective thermodynamic equilibrium.
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Photonic crystals-a class of materials whose optical properties derive from their structure in addition to their composition-can be created by self-assembling particles whose sizes are comparable to the wavelengths of visible light. Proof-of-principle studies have shown that DNA can be used to guide the self-assembly of micrometer-sized colloidal particles into fully programmable crystal structures with photonic properties in the visible spectrum. However, the extremely temperature-sensitive kinetics of micrometer-sized DNA-functionalized particles has frustrated attempts to grow large, monodisperse crystals that are required for photonic metamaterial applications. Here we describe a robust two-step protocol for self-assembling single-domain crystals that contain millions of optical-scale DNA-functionalized particles: Monodisperse crystals are initially assembled in monodisperse droplets made by microfluidics, after which they are grown to macroscopic dimensions via seeded diffusion-limited growth. We demonstrate the generality of our approach by assembling different macroscopic single-domain photonic crystals with metamaterial properties, like structural coloration, that depend on the underlying crystal structure. By circumventing the fundamental kinetic traps intrinsic to crystallization of optical-scale DNA-coated colloids, we eliminate a key barrier to engineering photonic devices from DNA-programmed materials.
Asunto(s)
Óptica y Fotónica , Fotones , Coloides/química , Cristalización , ADNRESUMEN
We introduce a method for solving the "inverse" phase equilibria problem: How should the interactions among a collection of molecular species be designed in order to achieve a target phase diagram? Using techniques from convex optimization theory, we show how to solve this problem for phase diagrams containing a large number of components and many coexisting phases with prescribed compositions. We apply our approach to commonly used mean-field models of multicomponent fluids and then use molecular simulations to verify that the designed interactions result in the target phase diagrams. Our approach enables the rational design of "programmable" fluids, such as biopolymer and colloidal mixtures, with complex phase behavior.
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Biomolecular condensates constitute a newly recognized form of spatial organization in living cells. Although many condensates are believed to form as a result of phase separation, the physicochemical properties that determine the phase behavior of heterogeneous biomolecular mixtures are only beginning to be explored. Theory and simulation provide invaluable tools for probing the relationship between molecular determinants, such as protein and RNA sequences, and the emergence of phase-separated condensates in such complex environments. This review covers recent advances in the prediction and computational design of biomolecular mixtures that phase-separate into many coexisting phases. First, we review efforts to understand the phase behavior of mixtures with hundreds or thousands of species using theoretical models and statistical approaches. We then describe progress in developing analytical theories and coarse-grained simulation models to predict multiphase condensates with the molecular detail required to make contact with biophysical experiments. We conclude by summarizing the challenges ahead for modeling the inhomogeneous spatial organization of biomolecular mixtures in living cells.
Asunto(s)
Proteínas , Proteínas/química , Simulación por ComputadorRESUMEN
Unlike fluids at thermal equilibrium, biomolecular mixtures in living systems can sustain nonequilibrium steady states, in which active processes modify the conformational states of the constituent molecules. Despite qualitative similarities between liquid-liquid phase separation in these systems, the extent to which the phase-separation kinetics differ remains unclear. Here we show that inhomogeneous chemical reactions can alter the nucleation kinetics of liquid-liquid phase separation in a manner that is consistent with classical nucleation theory, but can only be rationalized by introducing a nonequilibrium interfacial tension. We identify conditions under which nucleation can be accelerated without changing the energetics or supersaturation, thus breaking the correlation between fast nucleation and strong driving forces that is typical of phase separation and self-assembly at thermal equilibrium.
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We present a theoretical model for predicting the phase behavior of polymer solutions in which phase separation competes with oligomerization. Specifically, we consider scenarios in which the assembly of polymer chains into stoichiometric complexes prevents the chains from phase-separating via attractive polymer-polymer interactions. Combining statistical associating fluid theory with a two-state description of self-assembly, we find that this model exhibits rich phase behavior, including reentrance, and we show how system-specific phase diagrams can be derived graphically. Importantly, we discuss why these phase diagrams can resemble-and yet are qualitatively distinct from-phase diagrams of polymer solutions with lower critical solution temperatures.
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DNA-coated colloids can self-assemble into an incredible diversity of crystal structures, but their applications have been limited by poor understanding and control over the crystallization dynamics. To address this challenge, we use microfluidics to quantify the kinetics of DNA-programmed self-assembly along the entire crystallization pathway, from thermally activated nucleation through reaction-limited and diffusion-limited phases of crystal growth. Our detailed measurements of the temperature and concentration dependence of the kinetics at all stages of crystallization provide a stringent test of classical theories of nucleation and growth. After accounting for the finite rolling and sliding rates of micrometer-sized DNA-coated colloids, we show that modified versions of these classical theories predict the absolute nucleation and growth rates with quantitative accuracy. We conclude by applying our model to design and demonstrate protocols for assembling large single crystals with pronounced structural coloration, an essential step in creating next-generation optical metamaterials from colloids.
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Biomolecular condensates self-assemble when proteins and nucleic acids spontaneously demix to form droplets within the crowded intracellular milieu. This simple mechanism underlies the formation of a wide variety of membraneless compartments in living cells. To understand how multiple condensates with distinct compositions can self-assemble in such a heterogeneous system, which may not be at thermodynamic equilibrium, we study a minimal model in which we can "program" the pairwise interactions among hundreds of species. We show that the number of distinct condensates that can be reliably assembled grows superlinearly with the number of species in the mixture when the condensates share components. Furthermore, we show that we can predict the maximum number of distinct condensates in a mixture without knowing the details of the pairwise interactions. Simulations of condensate growth confirm these predictions and suggest that the physical rules governing the achievable complexity of condensate-mediated spatial organization are broadly applicable to biomolecular mixtures.
Asunto(s)
Modelos Químicos , Ácidos Nucleicos/química , Proteínas/químicaRESUMEN
Atomistic simulations can provide valuable, experimentally-verifiable insights into protein folding mechanisms, but existing ab initio simulation methods are restricted to only the smallest proteins due to severe computational speed limits. The folding of larger proteins has been studied using native-centric potential functions, but such models omit the potentially crucial role of non-native interactions. Here, we present an algorithm, entitled DBFOLD, which can predict folding pathways for a wide range of proteins while accounting for the effects of non-native contacts. In addition, DBFOLD can predict the relative rates of different transitions within a protein's folding pathway. To accomplish this, rather than directly simulating folding, our method combines equilibrium Monte-Carlo simulations, which deploy enhanced sampling, with unfolding simulations at high temperatures. We show that under certain conditions, trajectories from these two types of simulations can be jointly analyzed to compute unknown folding rates from detailed balance. This requires inferring free energies from the equilibrium simulations, and extrapolating transition rates from the unfolding simulations to lower, physiologically-reasonable temperatures at which the native state is marginally stable. As a proof of principle, we show that our method can accurately predict folding pathways and Monte-Carlo rates for the well-characterized Streptococcal protein G. We then show that our method significantly reduces the amount of computation time required to compute the folding pathways of large, misfolding-prone proteins that lie beyond the reach of existing direct simulation. Our algorithm, which is available online, can generate detailed atomistic models of protein folding mechanisms while shedding light on the role of non-native intermediates which may crucially affect organismal fitness and are frequently implicated in disease.
Asunto(s)
Algoritmos , Pliegue de Proteína , Proteínas Bacterianas/química , Biología Computacional , Cinética , Simulación de Dinámica Molecular/estadística & datos numéricos , Método de Montecarlo , Conformación Proteica , Desplegamiento Proteico , Programas Informáticos , Temperatura , TermodinámicaRESUMEN
Cotranslational folding depends on the folding speed and stability of the nascent protein. It remains difficult, however, to predict which proteins cotranslationally fold. Here, we simulate evolution of model proteins to investigate how native structure influences evolution of cotranslational folding. We developed a model that connects protein folding during and after translation to cellular fitness. Model proteins evolved improved folding speed and stability, with proteins adopting one of two strategies for folding quickly. Low contact order proteins evolve to fold cotranslationally. Such proteins adopt native conformations early on during the translation process, with each subsequently translated residue establishing additional native contacts. On the other hand, high contact order proteins tend not to be stable in their native conformations until the full chain is nearly extruded. We also simulated evolution of slowly translating codons, finding that slower translation speeds at certain positions enhances cotranslational folding. Finally, we investigated real protein structures using a previously published data set that identified evolutionarily conserved rare codons in Escherichia coli genes and associated such codons with cotranslational folding intermediates. We found that protein substructures preceding conserved rare codons tend to have lower contact orders, in line with our finding that lower contact order proteins are more likely to fold cotranslationally. Our work shows how evolutionary selection pressure can cause proteins with local contact topologies to evolve cotranslational folding.
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Biosíntesis de Proteínas , Pliegue de Proteína , Codón , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMEN
Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.
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Gránulos Citoplasmáticos/fisiología , Estructuras Citoplasmáticas/fisiología , Mapas de Interacción de Proteínas/fisiología , Fenómenos Biofísicos , Línea Celular Tumoral , Citoplasma/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Extracción Líquido-Líquido/métodos , Orgánulos/química , ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/fisiologíaRESUMEN
Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas Intrínsecamente Desordenadas/química , Pliegue de Proteína , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Cinética , Simulación de Dinámica Molecular , Fosfotransferasas/química , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Biosíntesis de Proteínas , Proteína Metiltransferasas/química , Proteína Metiltransferasas/genética , Proteína Metiltransferasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
A central goal of protein-folding theory is to predict the stochastic dynamics of transition paths-the rare trajectories that transit between the folded and unfolded ensembles-using only thermodynamic information, such as a low-dimensional equilibrium free-energy landscape. However, commonly used one-dimensional landscapes typically fall short of this aim, because an empirical coordinate-dependent diffusion coefficient has to be fit to transition-path trajectory data in order to reproduce the transition-path dynamics. We show that an alternative, first-principles free-energy landscape predicts transition-path statistics that agree well with simulations and single-molecule experiments without requiring dynamical data as an input. This "topological configuration" model assumes that distinct, native-like substructures assemble on a time scale that is slower than native-contact formation but faster than the folding of the entire protein. Using only equilibrium simulation data to determine the free energies of these coarse-grained intermediate states, we predict a broad distribution of transition-path transit times that agrees well with the transition-path durations observed in simulations. We further show that both the distribution of finite-time displacements on a one-dimensional order parameter and the ensemble of transition-path trajectories generated by the model are consistent with the simulated transition paths. These results indicate that a landscape based on transient folding intermediates, which are often hidden by one-dimensional projections, can form the basis of a predictive model of protein-folding transition-path dynamics.
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Pliegue de Proteína , Proteínas/química , Termodinámica , Difusión , Procesos EstocásticosRESUMEN
Despite considerable efforts, no physical mechanism has been shown to explain N-terminal codon bias in prokaryotic genomes. Using a systematic study of synonymous substitutions in two endogenous E. coli genes, we show that interactions between the coding region and the upstream Shine-Dalgarno (SD) sequence modulate the efficiency of translation initiation, affecting both intracellular mRNA and protein levels due to the inherent coupling of transcription and translation in E. coli. We further demonstrate that far-downstream mutations can also modulate mRNA levels by occluding the SD sequence through the formation of non-equilibrium secondary structures. By contrast, a non-endogenous RNA polymerase that decouples transcription and translation largely alleviates the effects of synonymous substitutions on mRNA levels. Finally, a complementary statistical analysis of the E. coli genome specifically implicates avoidance of intra-molecular base pairing with the SD sequence. Our results provide general physical insights into the coding-level features that optimize protein expression in prokaryotes.
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Codón Iniciador , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Motivos de Nucleótidos , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
To optimize a self-assembly reaction, it is essential to understand the factors that govern its pathway. Here, we examine the influence of nucleation pathways in a model system for addressable, multicomponent self-assembly based on a prototypical "DNA-brick" structure. By combining temperature-dependent dynamic light scattering and atomic force microscopy with coarse-grained simulations, we show how subtle changes in the nucleation pathway profoundly affect the yield of the correctly formed structures. In particular, we can increase the range of conditions over which self-assembly occurs by using stable multisubunit clusters that lower the nucleation barrier for assembling subunits in the interior of the structure. Consequently, modifying only a small portion of a structure is sufficient to optimize its assembly. Due to the generality of our coarse-grained model and the excellent agreement that we find with our experimental results, the design principles reported here are likely to apply generically to addressable, multicomponent self-assembly.
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ADN/química , Luz , Modelos Químicos , Dispersión de RadiaciónRESUMEN
Recent experiments and simulations have demonstrated that proteins can fold on the ribosome. However, the extent and generality of fitness effects resulting from cotranslational folding remain open questions. Here we report a genome-wide analysis that uncovers evidence of evolutionary selection for cotranslational folding. We describe a robust statistical approach to identify loci within genes that are both significantly enriched in slowly translated codons and evolutionarily conserved. Surprisingly, we find that domain boundaries can explain only a small fraction of these conserved loci. Instead, we propose that regions enriched in slowly translated codons are associated with cotranslational folding intermediates, which may be smaller than a single domain. We show that the intermediates predicted by a native-centric model of cotranslational folding account for the majority of these loci across more than 500 Escherichia coli proteins. By making a direct connection to protein folding, this analysis provides strong evidence that many synonymous substitutions have been selected to optimize translation rates at specific locations within genes. More generally, our results indicate that kinetics, and not just thermodynamics, can significantly alter the efficiency of self-assembly in a biological context.