RESUMO
The Corona pandemic has painfully taught us the threat of new pathogens in a globalized world and how vital modern vaccines are. Platform technologies play an important role in the discovery of new vaccines as reducing the time for the development dramatically - time that saves lives. Here, we present the protein Dodecin and how it may be utilized as a versatile platform technology to produce cheap and robust new vaccines for everyone in all parts of the world.
RESUMO
Step-by-step to great diversity: With the potential to synthesize multi-millions of bioactive compounds, modular polyketide synthases (PKSs) are of great importance. In this Highlight, new developments in the understanding of the structure and function of these proteins are reviewed.
Assuntos
Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Animais , Humanos , Modelos MolecularesRESUMO
Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-selective incorporation of fluorine in complex molecules have improved in recent years, approaches for the biosynthetic incorporation of fluorine in natural compounds are still rare. Here, we report a strategy to introduce fluorine into complex polyketides during biosynthesis. We exchanged the native acyltransferase domain of a polyketide synthase, which acts as the gatekeeper for the selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase. The resulting polyketide-synthase/fatty-acid-synthase hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl units in polyketide scaffolds. We demonstrate the feasibility of our approach in the chemoenzymatic synthesis of fluorinated 12- and 14-membered macrolactones and fluorinated derivatives of the macrolide antibiotics YC-17 and methymycin.
Assuntos
Policetídeos , Aciltransferases , Animais , Coenzima A , Flúor , Policetídeo SintasesRESUMO
De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the Type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT-malonyl-/acetyltransferase) and the condensation (KS-ß-ketoacyl synthase) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate-binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.
Assuntos
Caprilatos/química , Ácido Graxo Sintase Tipo I/química , Animais , Sítios de Ligação , Caprilatos/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Cinética , Camundongos , Modelos Moleculares , Estrutura MolecularRESUMO
Transmission electron cryo-microscopy (cryoEM) of vitrified biological specimens is a powerful tool for structural biology. Current preparation of vitrified biological samples starts off with sample isolation and purification, followed by the fixation in a freestanding layer of amorphous ice. Here, we demonstrate that ultrathin (â¼10 nm) smart molecular nanosheets having specific biorecognition sites embedded in a biorepulsive layer covalently bound to a mechanically stable carbon nanomembrane allow for a much simpler isolation and structural analysis. We characterize in detail the engineering of these nanosheets and their biorecognition properties employing complementary methods such as X-ray photoelectron and infrared spectroscopy, atomic force microscopy as well as surface plasmon resonance measurements. The desired functionality of the developed nanosheets is demonstrated by in situ selection of a His-tagged protein from a mixture and its subsequent structural analysis by cryoEM.
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Carbono , Elétrons , Microscopia Crioeletrônica , Microscopia de Força Atômica , Microscopia Eletrônica de TransmissãoRESUMO
Modularity is a fundamental property of megasynthases such as polyketide synthases (PKSs). In this study, we exploit the close resemblance between PKSs and animal fatty acid synthase (FAS) to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by sequence and structural information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel re-engineered modules resemble all four common types of PKSs and demonstrate that this approach can be a powerful tool to deliver products with higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design.
Assuntos
Ácido Graxo Sintase Tipo I/química , Policetídeo Sintases/química , Engenharia de Proteínas , Dobramento de Proteína , Ácido Graxo Sintase Tipo I/genética , Humanos , Policetídeo Sintases/genéticaRESUMO
Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection.
Assuntos
Proteínas Arqueais/química , Proteínas Quimiotáticas Aceptoras de Metil/química , Pyrococcus horikoshii/química , Archaea/química , Archaea/genética , Archaea/metabolismo , Quimiotaxia/genética , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Flagelos/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/biossíntese , Proteínas Quimiotáticas Aceptoras de Metil/genética , Conformação Proteica , Multimerização Proteica , Pyrococcus horikoshii/genética , Pyrococcus horikoshii/metabolismo , Transdução de SinaisRESUMO
The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.
Assuntos
Aminoácidos/genética , Códon de Terminação/genética , Ácido Graxo Sintase Tipo I/genética , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Aminoácidos/química , Animais , Códon de Terminação/química , Ácido Graxo Sintase Tipo I/química , Código Genético , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Modelos Moleculares , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Fatty acid synthases (FASs) and polyketide synthases (PKSs) condense acyl compounds to fatty acids and polyketides, respectively. Both, FASs and PKSs, harbor acyltransferases (ATs), which select substrates for condensation by ß-ketoacyl synthases (KSs). Here, we present the structural and functional characterization of the polyspecific malonyl/acetyltransferase (MAT) of murine FAS. We assign kinetic constants for the transacylation of the native substrates, acetyl- and malonyl-CoA, and demonstrate the promiscuity of FAS to accept structurally and chemically diverse CoA-esters. X-ray structural data of the KS-MAT didomain in a malonyl-loaded state suggests a MAT-specific role of an active site arginine in transacylation. Owing to its enzymatic properties and its accessibility as a separate domain, MAT of murine FAS may serve as versatile tool for engineering PKSs to provide custom-tailored access to new polyketides that can be applied in antibiotic and antineoplastic therapy.
Assuntos
Descoberta de Drogas , Ácido Graxo Sintase Tipo I , Policetídeo Sintases/metabolismo , Engenharia de Proteínas/métodos , Transferases , Acilação , Aciltransferases/química , Animais , Malonil Coenzima A , Camundongos , Policetídeos/síntese químicaRESUMO
To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution.