540 Chemie und zugeordnete Wissenschaften
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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).
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.
Modular polyketide synthases (PKSs) produce complex, bioactive secondary metabolites in assembly line-like multistep reactions. Longstanding efforts to produce novel, biologically active compounds by recombining intact modules to new modular PKSs have mostly resulted in poorly active chimeras and decreased product yields. Recent findings demonstrate that the low efficiencies of modular chimeric PKSs also result from rate limitations in the transfer of the growing polyketide chain across the non-cognate module:module interface and further processing of the non-native polyketide substrate by the ketosynthase (KS) domain. In this study, we aim at disclosing and understanding the low efficiency of chimeric modular PKSs and at establishing guidelines for modular PKSs engineering. To do so, we work with a bimodular PKS testbed and systematically vary substrate specificity, substrate identity, and domain:domain interfaces of the KS involved reactions. We observe that KS domains employed in our chimeric bimodular PKSs are bottlenecks with regards to both substrate specificity as well as interaction with the ACP. Overall, our systematic study can explain in quantitative terms why early oversimplified engineering strategies based on the plain shuffling of modules mostly failed and why more recent approaches show improved success rates. We moreover identify two mutations of the KS domain that significantly increased turnover rates in chimeric systems and interpret this finding in mechanistic detail.