RESUMEN
The major energy-producing reactions of biochemistry occur at biological membranes. Computational protein design now provides the opportunity to elucidate the underlying principles of these processes and to construct bioenergetic pathways on our own terms. Here, we review recent achievements in this endeavour of 'synthetic bioenergetics', with a particular focus on new enabling tools that facilitate the computational design of biocompatible de novo integral membrane proteins. We use recent examples to showcase some of the key computational approaches in current use and highlight that the overall philosophy of 'surface-swapping' - the replacement of solvent-facing residues with amino acids bearing lipid-soluble hydrophobic sidechains - is a promising avenue in membrane protein design. We conclude by highlighting outstanding design challenges and the emerging role of AI in sequence design and structure ideation.
Asunto(s)
Metabolismo Energético , Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Biología Computacional/métodos , Ingeniería de Proteínas/métodos , Modelos Moleculares , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to delineate the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.
Asunto(s)
Hemo , Oxidación-Reducción , Hemo/química , Hemo/metabolismo , Solubilidad , Agua/química , Agua/metabolismo , Citocromos/química , Citocromos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Electricidad Estática , Ingeniería de ProteínasRESUMEN
Sponges (phylum Porifera) harbour specific microbial communities that drive the ecology and evolution of the host. Understanding the structure and dynamics of these communities is emerging as a primary focus in marine microbial ecology research. Much of the work to date has focused on sponges from warm and shallow coastal waters, while sponges from the deep ocean remain less well studied. Here, we present a metataxonomic analysis of the microbial consortia associated with 23 individual deep-sea sponges. We identify a high abundance of archaea relative to bacteria across these communities, with certain sponge microbiomes comprising more than 90â% archaea. Specifically, the archaeal family Nitrosopumilaceae is prolific, comprising over 99â% of all archaeal reads. Our analysis revealed that sponge microbial communities reflect the host sponge phylogeny, indicating a key role for host taxonomy in defining microbiome composition. Our work confirms the contribution of both evolutionary and environmental processes to the composition of microbial communities in deep-sea sponges.
Asunto(s)
Archaea , Bacterias , Microbiota , Filogenia , Poríferos , Poríferos/microbiología , Archaea/clasificación , Archaea/genética , Archaea/aislamiento & purificación , Animales , Océano Atlántico , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Agua de Mar/microbiología , ARN Ribosómico 16S/genética , BiodiversidadRESUMEN
The deep sea is known to host novel bacteria with the potential to produce a diverse array of undiscovered natural products. Thus, understanding these bacteria is of broad interest in ecology and could also underpin applied drug discovery, specifically in the area of antimicrobials. Here, we isolate a new strain of Streptomyces from the tissue of the deep-sea sponge Polymastia corticata collected at a depth of 1869 m from the Gramberg Seamount in the Atlantic Ocean. This strain, which was given the initial designation A15ISP2-DRY2T, has a genome size of 9.29 Mb with a G+C content of 70.83âmol%. Phylogenomics determined that A15ISP2-DRY2T represents a novel species within the genus Streptomyces as part of the Streptomyces aurantiacus clade. The biosynthetic potential of A15ISP2-DRY2T was assessed relative to other members of the S. aurantiacus clade via comparative gene cluster family (GCF) analysis. This revealed a clear congruent relationship between phylogeny and GCF content. A15ISP2-DRY2T contains six unique GCFs absent elsewhere in the clade. Culture-based assays were used to demonstrate the antibacterial activity of A15ISP2-DRY2T against two drug-resistant human pathogens. Thus, we determine A15ISP2-DRY2T to be a novel bacterial species with considerable biosynthetic potential and propose the systematic name 'Streptomyces ortus' sp. nov.
Asunto(s)
Poríferos , Streptomyces , Streptomyces/química , Streptomyces/clasificación , Streptomyces/aislamiento & purificación , Agua de Mar/microbiología , Microbiología del Agua , Poríferos/microbiología , Animales , Composición de Base , Genoma BacterianoRESUMEN
Heme-containing integral membrane proteins are at the heart of many bioenergetic complexes and electron transport chains. The importance of these electron relay hubs across biology has inspired the design of de novo proteins that recreate their core features within robust, versatile, and tractable protein folds. To this end, we report here the computational design and in-cell production of a minimal diheme membrane cytochrome which successfully integrates into the cellular membrane of live bacteria. This synthetic construct emulates a four-helix bundle found in modern respiratory complexes but has no sequence homology to any polypeptide sequence found in nature. The two b-type hemes, which appear to be recruited from the endogenous heme pool, have distinct split redox potentials with values close to those of natural membrane-spanning cytochromes. The purified protein can engage in rapid biomimetic electron transport with small molecules, with other redox proteins, and with biologically relevant diffusive electron carriers. We thus report an artificial membrane metalloprotein with the potential to serve as a functional electron transfer module in both synthetic protocells and living systems.
Asunto(s)
Citocromos , Metaloproteínas , Citocromos/metabolismo , Oxidación-Reducción , Transporte de Electrón , Metaloproteínas/metabolismo , Hemo/metabolismoRESUMEN
After publication of this article [...].
RESUMEN
Coccolithophores are an important group of calcifying marine phytoplankton. Although coccolithophores are not silicified, some species exhibit a requirement for Si in the calcification process. These species also possess a novel protein (SITL) that resembles the SIT family of Si transporters found in diatoms. However, the nature of Si transport in coccolithophores is not yet known, making it difficult to determine the wider role of Si in coccolithophore biology. Here, we show that coccolithophore SITLs act as Na+ -coupled Si transporters when expressed in heterologous systems and exhibit similar characteristics to diatom SITs. We find that CbSITL from Coccolithus braarudii is transcriptionally regulated by Si availability and is expressed in environmental coccolithophore populations. However, the Si requirement of C. braarudii and other coccolithophores is very low, with transport rates of exogenous Si below the level of detection in sensitive assays of Si transport. As coccoliths contain only low levels of Si, we propose that Si acts to support the calcification process, rather than forming a structural component of the coccolith itself. Si is therefore acting as a micronutrient in coccolithophores and natural populations are only likely to experience Si limitation in circumstances where dissolved silicon (DSi) is depleted to extreme levels.
Asunto(s)
Diatomeas , Haptophyta , Silicio/metabolismo , Fitoplancton/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Diatomeas/genética , Diatomeas/metabolismo , Calcificación Fisiológica , Haptophyta/genética , Haptophyta/metabolismoRESUMEN
Diatoms are an important group of algae that can produce intricate silicified cell walls (frustules). The complex process of silicification involves a set of enigmatic integral membrane proteins that are thought to actively transport the soluble precursor of biosilica, dissolved silicic acid. Full-length silicic acid transporters are found widely across the diatoms while homologous shorter proteins have now been identified in a range of other organisms. It has been suggested that modern silicic acid transporters arose from the union of such partial sequences. Here, we present a computational study of the silicic acid transporters and related transporter-like sequences to help understand the structure, function and evolution of this class of membrane protein. The AlphaFold software predicts that all of the protein sequences studied here share a common fold in the membrane domain which is entirely different from the predicted folds of non-homologous silicic acid transporters from plants. Substrate docking reveals how conserved polar residues could interact with silicic acid at a central solvent-accessible binding site, consistent with an alternating access mechanism of transport. The structural conservation between these proteins supports a model where modern silicon transporters evolved from smaller ancestral proteins by gene fusion.
Asunto(s)
Diatomeas , Ácido Silícico , Ácido Silícico/química , Ácido Silícico/metabolismo , Diatomeas/genética , Diatomeas/química , Diatomeas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Silicio/química , Proteínas de la Membrana/metabolismo , Simulación por ComputadorRESUMEN
Tetrapyrrole cofactors such as heme and chlorophyll imprint their intrinsic reactivity and properties on a multitude of natural proteins and enzymes, and there is much interest in exploiting their functional and catalytic capabilities within minimal, de novo designed protein scaffolds. Here we describe how, using only natural biosynthetic and post-translational modification pathways, de novo designed soluble and hydrophobic proteins can be equipped with tetrapyrrole cofactors within living Escherichia coli cells. We provide strategies to achieve covalent and non-covalent heme incorporation within the de novo proteins and describe how the heme biosynthetic pathway can be co-opted to produce the light sensitive zinc protoporphyrin IX for loading into proteins in vivo. In addition, we describe the imaging of hydrophobic proteins and cofactor-rich protein droplets by electron and fluorescence microscopy, and how cofactors can be stripped from the de novo proteins to aid in vitro identification.
Asunto(s)
Proteínas/metabolismo , Clorofila , Escherichia coli/genética , Hemo , Proteínas/genética , TetrapirrolesRESUMEN
Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n-dodecyl-ß-d-maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family.
Asunto(s)
Proteínas Bacterianas , Expresión Génica , Haemophilus influenzae/genética , Proteínas de Transporte de Membrana , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Haemophilus influenzae/enzimología , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The stable isotopic composition of diatom silica is used as a proxy for nutrient utilisation in natural waters. This approach provides essential insight into the current and historic links between biological production, carbon cycling and climate. However, estimates of isotopic fractionation during diatom silica production from both laboratory and field studies are variable, and the biochemical pathways responsible remain unknown. Here, we investigate silicon isotopic fractionation through a series of chemical precipitation experiments that are analogous to the first stages of intracellular silica formation within the diatom silicon deposition vesicle. The novelty of our experiment is the inclusion of the R5 peptide, which is closely related to a natural biomolecule known to play a role in diatom silicification. Our results suggest that the presence of R5 induces a systematic but non-significant difference in fractionation behaviour. It thus appears that silicon isotopic fractionation in vitro is largely driven by an early kinetic fractionation during rapid precipitation that correlates with the initial amount of dissolved silica in the system. Our findings raise the question of how environmental changes might impact silicon isotopic fractionation in diatoms, and whether frustule archives record information in addition to silica consumption in surface water.
Asunto(s)
Biomimética , Precipitación Química , Isótopos/química , Dióxido de Silicio/química , CinéticaRESUMEN
To tackle the growing problem of antibiotic resistance, it is essential to identify new bioactive compounds that are effective against resistant microbes and safe to use. Natural products and their derivatives are, and will continue to be, an important source of these molecules. Sea sponges harbour a diverse microbiome that co-exists with the sponge, and these bacterial communities produce a rich array of bioactive metabolites for protection and resource competition. For these reasons, the sponge microbiota constitutes a potential source of clinically relevant natural products. To date, efforts in bioprospecting for these compounds have focused predominantly on sponge specimens isolated from shallow water, with much still to be learned about samples from the deep sea. Here we report the isolation of a new Micromonospora strain, designated 28ISP2-46T, recovered from the microbiome of a mid-Atlantic deep-sea sponge. Whole-genome sequencing reveals the capacity of this bacterium to produce a diverse array of natural products, including kosinostatin and isoquinocycline B, which exhibit both antibiotic and antitumour properties. Both compounds were isolated from 28ISP2-46T fermentation broths and were found to be effective against a plethora of multidrug-resistant clinical isolates. This study suggests that the marine production of isoquinocyclines may be more widespread than previously supposed and demonstrates the value of targeting the deep-sea sponge microbiome as a source of novel microbial life with exploitable biosynthetic potential.
Asunto(s)
Antibacterianos/aislamiento & purificación , Microbiota , Micromonospora/aislamiento & purificación , Poríferos/microbiología , Animales , Antibacterianos/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Océano Atlántico , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Secuenciación Completa del GenomaRESUMEN
Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mutación , Secuencias de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/genética , Modelos Moleculares , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Protoporfirinas/metabolismoRESUMEN
The deep ocean is the largest habitat for life on Earth, though the microorganisms that occupy this unique environmental niche remain largely unexplored. Due to the significant logistical and operational challenges associated with accessing the deep ocean, bioprospecting programmes that seek to generate novel products from marine organisms have, to date, focused predominantly on samples recovered from shallow seas. For this reason, the deep ocean remains a largely untapped resource of novel microbiological life and associated natural products. Here we report the establishment of the Bristol Sponge Microbiome Collection (BISECT), a unique repository of deep-sea microorganisms and associated metabolites isolated from the microbiota of marine sponges, recovered from previously unsurveyed regions of the mid Atlantic Ocean, at depths of 0.3-3 km. An integrated biodiscovery pipeline comprising molecular, genetic, bioinformatic and analytical tools is also described, which is being applied to interrogate this collection. The potential of this approach is illustrated using data reporting our initial efforts to identify antimicrobial natural product lead compounds. Prospects for the use of BISECT to address allied pharmaceutical needs, along with mechanisms of access to the collection are also discussed.
RESUMEN
The construction of artificial membrane proteins from first principles is of fundamental interest and holds considerable promise for new biotechnologies. This review considers the potential advantages of adopting a strictly minimalist approach to the process of membrane protein design. As well as the practical benefits of miniaturisation and simplicity for understanding sequence-structure-function relationships, minimalism should also support the abstract conceptualisation of membrane proteins as modular components for synthetic biology. These ideas are illustrated with selected examples that focus upon α-helical membrane proteins, and which demonstrate how such minimalist membrane proteins might be integrated into living biosystems.
Asunto(s)
Proteínas de la Membrana/química , Biotecnología , Proteínas de la Membrana/metabolismo , Relación Estructura-Actividad , Biología SintéticaRESUMEN
The de novo design of integral membrane proteins remains a major challenge in protein chemistry. Here, we describe the bottom-up design of a genetically-encoded synthetic membrane protein comprising only four amino acids (L, S, G and W) in the transmembrane domains. This artificial sequence, which we call REAMP for recombinantly expressed artificial membrane protein, is a single chain of 133 residues arranged into four antiparallel membrane-spanning α-helices. REAMP was overexpressed in Escherichia coli and localized to the cytoplasmic membrane with the intended transmembrane topology. Recombinant REAMP could be extracted from the cell membrane in detergent micelles and was robust and stable in vitro, containing helical secondary structure consistent with the original design. Engineered mono- and bis-histidine residues in the membrane domain of REAMP were able to coordinate heme in vitro, in a manner reminiscent of natural b-type cytochromes. This binding shifted the electrochemical potential of the cofactor, producing a synthetic hemoprotein capable of nascent redox catalysis. These results show that a highly reduced set of amino acids is sufficient to mimic some key properties of natural proteins, and that cellular biosynthesis is a viable route for the production of minimal de novo membrane sequences.
Asunto(s)
Escherichia coli/química , Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Membrana Celular/química , Escherichia coli/genética , Escherichia coli/metabolismo , Hemo/metabolismo , Proteínas de la Membrana/química , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Understanding how molecules in self-assembled soft-matter nanostructures are organized is essential for improving the design of next-generation nanomaterials. Imaging these assemblies can be challenging and usually requires processing, e.g., staining or embedding, which can damage or obscure features. An alternative is to use bioinspired mineralization, mimicking how certain organisms use biomolecules to template mineral formation. Previously, we have reported the design and characterization of Self-Assembled peptide caGEs (SAGEs) formed from de novo peptide building blocks. In SAGEs, two complementary, 3-fold symmetric, peptide hubs combine to form a hexagonal lattice, which curves and closes to form SAGE nanoparticles. As hexagons alone cannot tile onto spheres, the network must also incorporate nonhexagonal shapes. While the hexagonal ultrastructure of the SAGEs has been imaged, these defects have not been observed. Here, we show that positively charged SAGEs biotemplate a thin, protective silica coating. Electron microscopy shows that these SiO2-SAGEs do not collapse, but maintain their 3D shape when dried. Atomic force microscopy reveals a network of hexagonal and irregular features on the SiO2-SAGE surface. The dimensions of these (7.2 nm ± 1.4 nm across, internal angles 119.8° ± 26.1°) are in accord with the designed SAGE network and with coarse-grained modeling of the SAGE assembly. The SiO2-SAGEs are permeable to small molecules (<2 nm), but not to larger biomolecules (>6 nm). Thus, bioinspired silicification offers a mild technique that preserves soft-matter nanoparticles for imaging, revealing structural details <10 nm in size, while also maintaining desirable properties, such as permeability to small molecules.
Asunto(s)
Péptidos/síntesis química , Dióxido de Silicio/química , Tamaño de la Partícula , Péptidos/química , Propiedades de SuperficieRESUMEN
The alcohol-O-acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl-coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol-O-acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl-CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium-chain-length acyl-CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Acetiltransferasas/aislamiento & purificación , Clonación Molecular , Cromatografía de Gases y Espectrometría de Masas , Proteínas/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
Diatoms are an important group of eukaryotic algae with a curious evolutionary innovation: they sheath themselves in a cell wall made largely of silica. The cellular machinery responsible for silicification includes a family of membrane permeases that recognize and actively transport the soluble precursor of biosilica, silicic acid. However, the molecular basis of silicic acid transport remains obscure. Here, we identify experimentally tractable diatom silicic acid transporter (SIT) homologues and study their structure and function in vitro, enabled by the development of a new fluorescence method for studying substrate transport kinetics. We show that recombinant SITs are Na(+)/silicic acid symporters with a 1:1 protein: substrate stoichiometry and KM for silicic acid of 20 µM. Protein mutagenesis supports the long-standing hypothesis that four conserved GXQ amino acid motifs are important in SIT function. This marks a step towards a detailed understanding of silicon transport with implications for biogeochemistry and bioinspired materials.