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ConspectusCentral to the quest of understanding the emergence of life is to uncover the role of metals, particularly iron, in shaping prebiotic chemistry. Iron, as the most abundant of the accessible transition metals on the prebiotic Earth, played a pivotal role in early biochemical processes and continues to be indispensable to modern biology. Here, we discuss our recent contributions to probing the plausibility of prebiotic complexes with iron, including heme and iron-sulfur clusters, in mediating chemistry beneficial to a protocell. Laboratory experiments and spectroscopic findings suggest plausible pathways, often facilitated by UV light, for the synthesis of heme and iron-sulfur clusters. Once formed, heme displays catalytic, peroxidase-like activity when complexed with amphiphiles. This activity could have been beneficial in two ways. First, heme could have catalytically removed a molecule (H2O2) that could have had degradative effects on a protocell. Second, heme could have helped in the synthesis of the building blocks of life by coupling the reduction of H2O2 with the oxidation of organic substrates. The necessity of amphiphiles to avoid the formation of inactive complexes of heme is telling, as the modern-day electron transport chain possesses heme embedded within a lipid membrane. Conversely, prebiotic iron-sulfur peptides have yet to be reported to partition into lipid membranes, nor have simple iron-sulfur peptides been found to be capable of participating in the synthesis of organic molecules. Instead, iron-sulfur peptides span a wide range of reduction potentials complementary to the reduction potentials of hemes. The reduction potential of iron-sulfur peptides can be tuned by the type of iron-sulfur cluster formed, e.g., [2Fe-2S] versus [4Fe-4S], or by the substitution of ligands to the metal center. Since iron-sulfur clusters easily form upon stochastic encounters between iron ions, hydrosulfide, and small organic molecules possessing a thiolate, including peptides, the likelihood of soluble iron-sulfur clusters seems to be high. What remains challenging to determine is if iron-sulfur peptides participated in early prebiotic chemistry or were recruited later when protocellular membranes evolved that were compatible with the exploitation of electron transfer for the storage of energy as a proton gradient. This problem mirrors in some ways the difficulty in deciphering the origins of metabolism as a whole. Chemistry that resembles some facets of extant metabolism must have transpired on the prebiotic Earth, but there are few clues as to how and when such chemistry was harnessed to support a (proto)cell. Ultimately, unraveling the roles of hemes and iron-sulfur clusters in prebiotic chemistry promises to deepen our understanding of the origins of life on Earth and aids the search for life elsewhere in the universe.
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Nicotinamide adenine dinucleotide (NAD+) is a redox active molecule that is universally found in biology. Despite the importance and simplicity of this molecule, few reports exist that investigate which molecular features are important for the activity of this ribodinucleotide. By exploiting the nonenzymatic reduction and oxidation of NAD+ by pyruvate and methylene blue, respectively, we were able to identify key molecular features necessary for the intrinsic activity of NAD+ through kinetic analysis. Such features may explain how NAD+ could have been selected early during the emergence of life. Simpler molecules, such as nicotinamide, that lack an anomeric carbon are incapable of accepting electrons from pyruvate. The phosphate moiety inhibits activity in the absence of metal ions but facilitates activity at physiological pH and model prebiotic conditions by recruiting catalytic Mg2+. Reduction proceeds through consecutive single electron transfer events. Of the derivatives tested, including nicotinamide mononucleotide, nicotinamide riboside, 3-(aminocarbonyl)-1-(2,3-dihydroxypropyl)pyridinium, 1-methylnicotinamide, and nicotinamide, only NAD+ and nicotinamide mononucleotide would be capable of efficiently accepting and donating electrons within a nonenzymatic electron transport chain. The data are consistent with early metabolic chemistry exploiting NAD+ or nicotinamide mononucleotide and not simpler molecules.
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Magnésio , NAD , Niacinamida , Oxirredução , NAD/química , NAD/metabolismo , Magnésio/química , Niacinamida/química , Niacinamida/análogos & derivados , Sítios de Ligação , Cinética , Mononucleotídeo de Nicotinamida/química , Mononucleotídeo de Nicotinamida/metabolismo , Compostos de Piridínio/química , Azul de Metileno/química , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Transporte de ElétronsRESUMO
Iron-sulfur clusters are thought to be ancient cofactors that could have played a role in early protometabolic systems. Thus far, redox active, prebiotically plausible iron-sulfur clusters have always contained cysteine ligands to the cluster. However, extant iron-sulfur proteins can be found to exploit other modes of binding, including ligation by histidine residues, as seen with [2Fe-2S] Rieske and MitoNEET proteins. Here, we investigated the ability of cysteine- and histidine-containing peptides to coordinate a mononuclear Fe2+ center and a [2Fe-2S] cluster and compare their properties with purified iron-sulfur proteins. The iron-sulfur peptides were characterized by UV-vis, circular dichroism, and paramagnetic NMR spectroscopies and cyclic voltammetry. Small (≤6 amino acids) peptides can coordinate [2Fe-2S] clusters through a combination of cysteine and histidine residues with similar reduction potentials as their corresponding proteins. Such complexes may have been important for early cell-like systems.
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Histidina , Proteínas Ferro-Enxofre , Cisteína/metabolismo , Histidina/química , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Peptídeos/metabolismo , Enxofre/metabolismoRESUMO
It is common to compare life with machines. Both consume fuel and release waste to run. In biology, the engine that drives the living system is referred to as metabolism. However, attempts at deciphering the origins of metabolism do not focus on this energetic relationship that sustains life but rather concentrate on nonenzymatic reactions that produce all the intermediates of an extant metabolic pathway. Such an approach is akin to studying the molecules produced from the burning of coal instead of deciphering how the released energy drives the movement of pistons and ultimately the train when investigating the mechanisms behind locomotion. Theories that do explicitly invoke geological chemical gradients to drive metabolism most frequently feature hydrothermal vent conditions, but hydrothermal vents are not the only regions of the early Earth that could have provided the fuel necessary to sustain the Earth's first (proto)cells. Here, we give examples of prior reports on protometabolism and highlight how more recent investigations of out-of-equilibrium systems may point to alternative scenarios more consistent with the majority of prebiotic chemistry data accumulated thus far. This article is part of the theme issue 'Emergent phenomena in complex physical and socio-technical systems: from cells to societies'.
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Fontes Hidrotermais , Planeta Terra , Fontes Hidrotermais/químicaRESUMO
The near universal availability of UV-Visible spectrophotometers makes this instrument a highly exploited tool for the inexpensive, rapid examination of iron-sulfur clusters. Yet, the analysis of iron-sulfur cluster reconstitution experiments by UV-Vis spectroscopy is notoriously difficult due to the presence of broad, ill-defined peaks. Other types of spectroscopies, such as electron paramagnetic resonance spectroscopy and Mössbauer spectroscopy, are superior in characterizing the type of cluster present and their associated electronic transitions but require expensive, less readily available equipment. Here, we describe a tool that utilizes the accessible and convenient platform of Microsoft Excel to allow for the semi-quantitative analysis of iron-sulfur clusters by UV-Vis spectroscopy. This tool, which we call Fit-FeS, could potentially be used to additionally decompose spectra of solutions containing chromophores other than iron-sulfur clusters.
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Ferro/química , Enxofre/química , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Ferrosos/química , Conformação Molecular , Biblioteca de Peptídeos , Peptídeos/química , Espectrofotometria UltravioletaRESUMO
Model protocells have long been constructed with fatty acids, because these lipids are prebiotically plausible and can, at least theoretically, support a protocell life cycle. However, fatty acid protocells are stable only within a narrow range of pH and metal ion concentration. This instability is particularly problematic as the early Earth would have had a range of conditions, and extant life is completely reliant on metal ions for catalysis and the folding and activity of biological polymers. Here, prebiotically plausible monoacyl cyclophospholipids are shown to form robust vesicles that survive a broad range of pH and high concentrations of Mg2+ , Ca2+ , and Na+ . Importantly, stability to Mg2+ and Ca2+ is improved by the presence of environmental concentrations of Na+ . These results suggest that cyclophospholipids, or lipids with similar characteristics, may have played a central role during the emergence of Darwinian evolution.
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Células Artificiais , Íons , Metais , Fosfolipídeos , Células Artificiais/efeitos dos fármacos , Catálise , Ácidos Graxos/química , Íons/química , Metais/química , Fosfolipídeos/farmacologiaRESUMO
Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.
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Aptâmeros de Nucleotídeos/química , Nanoestruturas/química , Proteínas/química , RNA/química , Aptâmeros de Nucleotídeos/genética , Sistema Livre de Células/metabolismo , Escherichia coli/genética , Expressão Gênica , Modelos Moleculares , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Proteínas/genética , RNA/genética , Estabilidade de RNA , Corantes de Rosanilina/química , Biologia Sintética/métodosRESUMO
Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron-sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe-2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions to disrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or L-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe-2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione-protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.
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Glutarredoxinas/química , Glutationa/química , Eletricidade Estática , Dicroísmo Circular , Humanos , Ligação de Hidrogênio , Ligantes , Estereoisomerismo , Compostos de Sulfidrila/químicaRESUMO
Antibacterial resistance is an issue of increasing severity as current antibiotics are losing their effectiveness and fewer antibiotics are being developed. New methods for combating bacterial virulence are required. Modulating molecular communication among bacteria can alter phenotype, including attachment to epithelia, biofilm formation, and even toxin production. Intercepting and modulating communication networks provide a means to attenuate virulence without directly interacting with the bacteria of interest. In this work, we target communication mediated by the quorum sensing (QS) bacterial autoinducer-2, AI-2. We have assembled a capsule of biological polymers alginate and chitosan, attached an AI-2 processing kinase, LsrK, and provided substrate, ATP, for enzymatic alteration of AI-2 in culture fluids. Correspondingly, AI-2 mediated QS activity is diminished. All components of this system are "biofabricated"-they are biologically derived and their assembly is accomplished using biological means. Initially, component quantities and kinetics were tested as assembled in microtiter plates. Subsequently, the identical components and assembly means were used to create the "artificial cell" capsules. The functionalized capsules, when introduced into populations of bacteria, alter the dynamics of the AI-2 bacterial communication, attenuating QS activated phenotypes. We envision the assembly of these and other capsules or similar materials, as means to alter QS activity in a biologically compatible manner and in many environments, including in humans.
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Células Artificiais/metabolismo , Biopolímeros/química , Proteínas de Escherichia coli/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Percepção de Quorum/genética , Proteínas Recombinantes/metabolismo , Alginatos/química , Células Artificiais/química , Quitosana/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Homosserina/análogos & derivados , Homosserina/química , Homosserina/metabolismo , Lactonas/química , Lactonas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmídeos/genética , Proteínas Recombinantes/genéticaRESUMO
A new R tool is described that rapidly identifies, ranks, and clusters sequence patterns coordinated to metallocofactors. This tool, PdPDB, fills a void because, unlike currently available tools, PdPDB searches through sequences with metal coordination as the primary determinant and can identify patterns consisting of amino acids, nucleotides, and small molecule ligands at once. PdPDB was tested by analyzing structures that coordinate Fe2+/3+, [2Fe-2S], [4Fe-4S], Zn2+, and Mg2+ cofactors. PdPDB confirmed previously identified sequence motifs and revealed which residues are enriched (e.g., glycine) and are under-represented (e.g., glutamine) near ligands to metal centers. The data show the similarities and differences between different metal-binding sites. The patterns that coordinate metallocofactors vary, depending upon whether the metal ions play a structural or catalytic role, with catalytic metal centers exhibiting partial coordination by small molecule ligands. PdPDB 2.0.1 is freely available as a CRAN package.
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Proteínas Ferro-Enxofre/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Aminoácidos/química , Aminoácidos/farmacologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Humanos , Proteínas Ferro-Enxofre/química , Ligantes , Nucleotídeos/química , Nucleotídeos/farmacologia , Ligação Proteica , SoftwareRESUMO
The selection of RNA and DNA aptamers now has a long history. However, the ability to directly select for conformational changes upon ligand binding has remained elusive. These difficulties have stymied attempts at making small molecule responsive strand displacement circuitry as well as synthetic riboswitches. Herein we present a detailed strand displacement based selection protocol to directly select for RNA molecules with switching activity. The library was based on a previously selected thiamine pyrophosphate riboswitch. The fully in vitro methodology gave sequences that showed strong strand displacement activity in the presence of thiamine pyrophosphate. Further, the selected sequences possessed riboswitch activity similar to that of natural riboswitches. The presented methodology should aid in the design of more complex, environmentally responsive strand displacement circuitry and in the selection of riboswitches responsive to toxic ligands.
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Aptâmeros de Nucleotídeos/genética , RNA/genética , Riboswitch/genética , Aptâmeros de Nucleotídeos/química , Ligantes , RNA/química , Bibliotecas de Moléculas Pequenas/química , Tiamina Pirofosfato/genéticaRESUMO
Model prebiotic dipeptide sequences were identified by bioinformatics and DFT and molecular dynamics calculations. The peptides were then synthesized and evaluated for metal affinity and specificity. Cysteine containing dipeptides were not associated with metal affinities that followed the Irving-Williams series but did follow the concentration trends found in seawater.
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The construction of genetically encoded cellular mimics in compartments containing organized synthetic cytosols is desirable for the development of artificial cells. Phase separated aqueous domains were placed within water-in-oil emulsion droplets in a manner compatible with transcription and translation machinery. Aqueous two-phase and three-phase systems (ATPS and A3PS) were assembled with dextran, poly(ethylene glycol), and Ficoll. Aqueous two-phase systems were capable of supporting the cell-free expression of protein within water droplets, whereas the aqueous three-phase-based system did not give rise to detectable protein synthesis. The expressed protein preferentially partitioned to the dextran-enriched phase. The system could serve as a foundation for building cellular mimics with liquid organelles.
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Óleos/química , Biossíntese de Proteínas , Transcrição Gênica , Água/química , Sistema Livre de Células/química , Dextranos/química , Ficoll/química , Polietilenoglicóis/químicaRESUMO
Contemporary phospholipid-based cell membranes are formidable barriers to the uptake of polar and charged molecules ranging from metal ions to complex nutrients. Modern cells therefore require sophisticated protein channels and pumps to mediate the exchange of molecules with their environment. The strong barrier function of membranes has made it difficult to understand the origin of cellular life and has been thought to preclude a heterotrophic lifestyle for primitive cells. Although nucleotides can cross dimyristoyl phosphatidylcholine membranes through defects formed at the gel-to-liquid transition temperature, phospholipid membranes lack the dynamic properties required for membrane growth. Fatty acids and their corresponding alcohols and glycerol monoesters are attractive candidates for the components of protocell membranes because they are simple amphiphiles that form bilayer membrane vesicles that retain encapsulated oligonucleotides and are capable of growth and division. Here we show that such membranes allow the passage of charged molecules such as nucleotides, so that activated nucleotides added to the outside of a model protocell spontaneously cross the membrane and take part in efficient template copying in the protocell interior. The permeability properties of prebiotically plausible membranes suggest that primitive protocells could have acquired complex nutrients from their environment in the absence of any macromolecular transport machinery; that is, they could have been obligate heterotrophs.
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Membrana Celular/metabolismo , Fenômenos Fisiológicos Celulares , Modelos Biológicos , Oligonucleotídeos/metabolismo , Transporte Biológico , Membrana Celular/química , Permeabilidade da Membrana Celular/fisiologia , Ácidos Graxos/metabolismo , Processos Heterotróficos , Nucleotídeos/metabolismo , Ribose/metabolismo , Moldes Genéticos , Fatores de TempoRESUMO
HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.
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Cisteína , Proteínas Fúngicas , Proteínas Ferro-Enxofre , Peptídeos , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Cisteína/metabolismo , Cisteína/química , Peptídeos/metabolismo , Peptídeos/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Ferro/metabolismo , Ligação Proteica , Análise Espectral Raman , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Prebiotic soup experiments have shown that the molecular building blocks of life can be built under prebiotically plausible conditions. From this starting point, researchers have launched continued studies of polymerization and explorations of the breadth of RNA function. Recently, effort has intensified to examine experimentally another stage of the origins of life: the assembly of the molecular parts into model protocells intended to represent the first primitive, cell-like systems to emerge on Earth. Although it may not be possible to recreate the precise sequence of events that led to cellular life, laboratory experiments have begun to show what was and was not possible. Prebiotically plausible lipid vesicles form easily and have many properties that are conducive to cellular function. In addition to protecting nascent replicating genetic systems from parasitic sequences, vesicles facilitate evolution. The data thus far suggest that prebiotically plausible vesicles could have grown, divided, and promoted competition between distinct chemical systems. Most protocellular studies to date have probed the role of self-replication, one feature of extant life in the emergence of the first cellular system. Undoubtedly replicating systems were crucial for protocellular evolution, but other features of life must have been important as well. For example, life does not exist in isolation. A living system must cope with and adapt to environmental fluctuations to survive. The protocell must have generated some of these fluctuations because cellular activity necessarily modifies its surroundings by selectively absorbing nutrients and releasing unwanted molecules. It seems likely that life would have faced this challenge early and either emerged in dynamic locales that continuously regenerated conditions conducive to life or exploited mechanisms to physically move to new areas not depleted in resources. Further studies that explore non-replication-based aspects of the origins of life could reveal a more complete picture of the transition from prebiotic chemistry to early life.
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Células Artificiais/metabolismo , Planeta Terra , Meio Ambiente , Modelos Biológicos , Origem da VidaRESUMO
Several laboratories are pursuing the synthesis of cellular systems from different directions, including those that begin with simple chemicals to those that exploit existing cells. The methods that begin with nonliving components tend to focus on mimicking specific features of life, such as genomic replication, protein synthesis, sensory systems, and compartment formation, growth, and division. Conversely, the more prevalent synthetic biology approaches begin with something that is already alive and seek to impart new behavior on existing cells. Here we discuss advances in building cell-like systems that mimic key features of life with defined components.
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Aerosols are abundant on the Earth and likely played a role in prebiotic chemistry. Aerosol particles coagulate, divide, and sample a wide variety of conditions conducive to synthesis. While much work has centered on the generation of aerosols and their chemistry, little effort has been expended on their fate after settling. Here, using a laboratory model, we show that aqueous aerosols transform into cell-sized protocellular structures upon entry into aqueous solution containing lipid. Such processes provide for a heretofore unexplored pathway for the assembly of the building blocks of life from disparate geochemical regions within cell-like vesicles with a lipid bilayer in a manner that does not lead to dilution. The efficiency of aerosol to vesicle transformation is high with prebiotically plausible lipids, such as decanoic acid and decanol, that were previously shown to be capable of forming growing and dividing vesicles. The high transformation efficiency with 10-carbon lipids in landing solutions is consistent with the surface properties and dynamics of short-chain lipids. Similar processes may be operative today as fatty acids are common constituents of both contemporary aerosols and the sea. Our work highlights a new pathway that may have facilitated the emergence of the Earth's first cells.
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There is currently no plausible path for the emergence of a self-replicating protocell, because prevalent formulations of model protocells are built with fatty acid vesicles that cannot withstand the concentrations of Mg2+ needed for the function and replication of nucleic acids. Although prebiotic chelates increase the survivability of fatty acid vesicles, the resulting model protocells are incapable of growth and division. Here, we show that protocells made of mixtures of cyclophospholipids and fatty acids can grow and divide in the presence of Mg2+-citrate. Importantly, these protocells retain encapsulated nucleic acids during growth and division, can acquire nucleotides from their surroundings, and are compatible with the nonenzymatic extension of an RNA oligonucleotide, chemistry needed for the replication of a primitive genome. Our work shows that prebiotically plausible mixtures of lipids form protocells that are active under the conditions necessary for the emergence of Darwinian evolution.