Universitätspublikationen
Refine
Year of publication
Document Type
- Article (23) (remove)
Has Fulltext
- yes (23)
Is part of the Bibliography
- no (23) (remove)
Keywords
- Biochemistry (2)
- Metabolic engineering (2)
- Proteins (2)
- multienzyme (2)
- protein engineering (2)
- 1-octanol (1)
- 3D reconstruction and image processing (1)
- Applied microbiology (1)
- Biocatalysis (1)
- Biofuel (1)
In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).
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.
Flavins are employed to transform physical input into biological output signals. In this function, flavins catalyze a variety of light-induced reactions and redox processes. However, nature also provides flavoproteins with the ability to uncouple the mediation of signals. Such proteins are the riboflavin-binding proteins (RfBPs) with their function to store riboflavin for fast delivery of FMN and FAD. Here we present in vitro and in vivo data showing that the recently discovered archaeal dodecin is an RfBP, and we reveal that riboflavin storage is not restricted to eukaryotes. However, the function of the prokaryotic RfBP dodecin seems to be adapted to the requirement of a monocellular organism. While in eukaryotes RfBPs are involved in trafficking riboflavin, and dodecin is responsible for the flavin homeostasis of the cell. Although only 68 amino acids in length, dodecin is of high functional versatility in neutralizing riboflavin to protect the cellular environment from uncontrolled flavin reactivity. Besides the predominant ultrafast quenching of excited states, dodecin prevents light-induced riboflavin reactivity by the selective degradation of riboflavin to lumichrome. Coordinated with the high affinity for lumichrome, the directed degradation reaction is neutral to the cellular environment and provides an alternative pathway for suppressing uncontrolled riboflavin reactivity. Intriguingly, the different structural and functional properties of a homologous bacterial dodecin suggest that dodecin has different roles in different kingdoms of life.
Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins.
Modularity is an aspect of a decomposable system with a coordinating authority that acts as a glue which holds the loosely held components. These multi-component entities (“modules”) facilitate rewiring into different designs allowing for change. Such modular character is a fundamental property of many biological entities, especially the family of megasynthases such as polyketide synthases (PKSs). The ability of these PKSs to produce diverse product spectra is strongly coupled to their broad architectural modularity. Decoding the molecular basis of modularity, i.e. identifying the folds and domains that comprise the modules as well as understanding constrains of the assembly of modules, is of utmost importance for harnessing megasynthases for the synthesis of designer compounds. In this study, we exploit the close semblance between PKSs and animal FAS to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by structural and sequence 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 engineered modules resemble all four common module types of PKSs and demonstrate that this approach can be a powerful tool to create 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.
Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1 Å resolution map of yeast FAS from 15 000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.
Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum.
Covering: mid 1990s to 2018
Over the last two decades, diverse approaches have been explored to generate new polyketides by engineering polyketide synthases (PKSs). Although it has been proven possible to produce new compounds by designed PKSs, engineering strategies failed to make polyketides available via widely applicable rules and protocols. Still, organic synthetic routes have to be employed whenever new polyketides are needed for applications in medicine, agriculture, and industry. In light of the rising demand for commodity products from feedstock and for fast and cheap access to pharmaceutical compounds, the need for harnessing PKSs to produce such molecules is more urgent than ever before. In this review, we focus on a multitude of approaches to engineer modular PKSs by swapping and replacing PKS modules and domains, which we analyze in the light of recent structural and biochemical data. We conclude with an outlook on possible strategies on how to increase success rates of PKS engineering in future.
The authors regret that there is an error present in the units displayed in the sentence “The dissociation constant of docking domains or modules connected by docking domains was found to be KD 70–130 mM (ref. 35) and KD 1–2 mM (ref. 59), respectively.” within Section 3.1. Module–module exchanges. The corrected version of this sentence is as follows:
The dissociation constant of docking domains or modules connected by docking domains was found to be KD 70–130 μM (ref. 35) and KD 1–2 mM (ref. 59), respectively.
The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.
Sustainable biosynthesis of chemicals and efforts to create new molecules of interest require efficient enzymes and pathways as well as comprehensive tools and technologies to implement this rewiring. Enzymes are the key components for construction of efficient biosynthetic pathways, enzyme characterization and engineering can help to identify key enzymes and regulatory factors for construction of biosynthetic pathways, as well as improve enzyme performance; Pathway engineering can help construct biosynthetic pathways and balance metabolic network to improve biosynthetic efficiency; Tools and technologies facilitate the engineering of enzymes, pathways, and whole cells. This special issue focusing on “Pathway and Protein Engineering for Biosynthesis” comprises eight review articles and nine original research articles, which highlight and showcase current progress on Pathway and Protein Engineering and their application for biosynthesis.