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The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Cheryl A Kerfeld – 1st expert on this subject based on the ideXlab platform
redox characterization of electrode immobilized Bacterial Microcompartment shell proteins engineered to bind metal centersACS Applied Bio Materials, 2020Co-Authors: Jefferson S Plegaria, Matthew D Yates, Sarah M Glaven, Cheryl A KerfeldAbstract:
Bacterial Microcompartment (BMC) shells are modular, selectively-permeable, nanoscale protein shells that self-assemble from hexagonal and pentagonal building blocks in vivo or in vitro. Natural and engineered BMC shells co-localize and concentrate catalysts and metabolites in their lumen, increasing reaction kinetics. Here, we describe the design and characterization of a shell protein (pseudohexameric/trimeric BMC-T1HO protein) engineered to coordinate a Cu ion in its pore. Several designs, each varying the position of an introduced coordinating histidine residue, were shown to maintain their trimeric oligomerization state upon Cu coordination via chemical denaturation experiments. We measured reversible redox activity from electrode-bound Cu-3His BMC-T1HO variants, with formal potential(s) that were dependent on the Cu coordination site within the discoidal shaped trimer (Eo’ = +208 to +265 mV vs. SHE). These results represent important steps toward expanding the functionality (Cu coordination) and app…
functionalization of Bacterial Microcompartment shell proteins with covalently attached hemeFrontiers in Bioengineering and Biotechnology, 2020Co-Authors: Jingcheng Huang, Cheryl A Kerfeld, Bryan Ferlez, Eric J Young, David M Kramer, Daniel C DucatAbstract:
Author(s): Huang, Jingcheng; Ferlez, Bryan H; Young, Eric J; Kerfeld, Cheryl A; Kramer, David M; Ducat, Daniel C | Abstract: Heme is a versatile redox cofactor that has considerable potential for synthetic biology and bioelectronic applications. The capacity to functionalize non-heme-binding proteins with covalently bound heme moieties in vivo could expand the variety of bioelectronic materials, particularly if hemes could be attached at defined locations so as to facilitate position-sensitive processes like electron transfer. In this study, we utilized the cytochrome maturation system I to develop a simple approach that enables incorporation of hemes into the backbone of target proteins in vivo. We tested our methodology by targeting the self-assembling Bacterial Microcompartment shell proteins, and inserting functional hemes at multiple locations in the protein backbone. We found substitution of three amino acids on the target proteins promoted heme attachment with high occupancy. Spectroscopic measurements suggested these modified proteins covalently bind low-spin hemes, with relative low redox midpoint potentials (about -210 mV vs. SHE). Heme-modified shell proteins partially retained their self-assembly properties, including the capacity to hexamerize, and form inter-hexamer attachments. Heme-bound shell proteins demonstrated the capacity to integrate into higher-order shell assemblies, however, the structural features of these macromolecular complexes was sometimes altered. Altogether, we report a versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins, and provide design considerations on how heme incorporation may interface with native assembly properties in engineered proteins.
a designed Bacterial Microcompartment shell with tunable composition and precision cargo loadingMetabolic Engineering, 2019Co-Authors: Bryan Ferlez, Markus Sutter, Cheryl A KerfeldAbstract:
Abstract Microbes often augment their metabolism by conditionally constructing proteinaceous organelles, known as Bacterial Microcompartments (BMCs), that encapsulate enzymes to degrade organic compounds or assimilate CO2. BMCs self-assemble and are spatially delimited by a semi-permeable shell made up of hexameric, trimeric, and pentameric shell proteins. Bioengineers aim to recapitulate the organization and efficiency of these complex biological architectures by redesigning the shell to incorporate non-native enzymes from biotechnologically relevant pathways. To meet this challenge, a diverse set of synthetic biology tools are required, including methods to manipulate the properties of the shell as well as target and organize cargo encapsulation. We designed and determined the crystal structure of a synthetic shell protein building block with an inverted sidedness of its N- and C-terminal residues relative to its natural counterpart; the inversion targets genetically fused protein cargo to the lumen of the shell. Moreover, the titer of fluorescent protein cargo encapsulated using this strategy is controllable using an inducible tetracycline promoter. These results expand the available set of building blocks for precision engineering of BMC-based nanoreactors and are compatible with orthogonal methods which will facilitate the installation and organization of multi-enzyme pathways.
Martin J Warren – 2nd expert on this subject based on the ideXlab platform
effect of metabolosome encapsulation peptides on enzyme activity coaggregation incorporation and Bacterial Microcompartment formationMicrobiologyOpen, 2020Co-Authors: Rokas Juodeikis, Stefanie Frank, Michael B Prentice, Judith Mantell, Ian R Brown, Paul Verkade, Derek N Woolfson, Matthias Mayer, Martin J WarrenAbstract:
Metabolosomes, catabolic Bacterial Microcompartments, are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multi-component protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation prior to the formation of the outer shell. We investigated the effect of three distinct encapsulation peptides on foreign cargo in a recombinant BMC system. Our data demonstrate that these peptides cause variation with respect to enzyme activity and protein aggregation. We observed that the level of protein aggregation generally correlates with the size of metabolosomes, while in the absence of cargo BMCs self-assemble into smaller compartments. The results agree with a flexible model for BMC formation based around the ability of the BMC shell to associate with an aggregate formed due to the interaction of encapsulation peptides.
Bacterial Microcompartment mediated ethanolamine metabolism in escherichia coli urinary tract infectionInfection and Immunity, 2019Co-Authors: Katherine Dadswell, Martin J Warren, Mingzhi Liang, Ian R Brown, Sinead Creagh, Edward Mccullagh, Alan Mcnally, John Macsharry, Michael B PrenticeAbstract:
ABSTRACT Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic Escherichia coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal E. coli, both pathogenic enterohemorrhagic E. coli and commensal strains. During a UTI, urease-negative E. coli bacteria thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTIs is understudied. We evaluated the role of the metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analyzed infected urine samples by culture, high-performance liquid chromatography, reverse transcription-quantitative PCR, and genomic sequencing. The ethanolamine concentration in urine was comparable to the concentration of the most abundant reported urinary amino acid, d-serine. Transcription of the eut operon was detected in the majority of urine samples containing E. coli screened. All sequenced UPEC strains had conserved eut operons, while metabolic genotypes previously associated with UTI (dsdCXA, metE) were mainly limited to phylogroup B2. In vitro ethanolamine was found to be utilized as a sole source of nitrogen by UPEC strains. The metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly, eutE (which encodes acetaldehyde dehydrogenase) was required for UPEC strains to utilize ethanolamine to gain a growth advantage in AUM, suggesting that ethanolamine is also utilized as a carbon source. These data suggest that urinary ethanolamine is a significant additional carbon and nitrogen source for infecting E. coli strains.
biotechnological advances in Bacterial Microcompartment technologyTrends in Biotechnology, 2019Co-Authors: David Palmer, Martin J WarrenAbstract:
Bacterial Microcompartments (BMCs) represent proteinaceous macromolecular nanobioreactors that are found in a broad range of bacteria, and which are associated with either anabolic or catabolic processes. They consist of a semipermeable outer shell that packages a central metabolic enzyme or pathway, providing both enhanced flux and protection against toxic intermediates. Recombinant production of BMCs has led to their repurposing with the incorporation of altogether new pathways. Deconstructing BMCs into their component parts has shown that some individual shell proteins self-associate into filaments that can be further modified into a cytoplasmic scaffold, or cytoscaffold, to which enzymes/proteins can be targeted. BMCs therefore represent a modular system that is highly suited for engineering biological systems for useful purposes.
Todd O Yeates – 3rd expert on this subject based on the ideXlab platform
molecular dynamics simulations of selective metabolite transport across the propanediol Bacterial Microcompartment shellJournal of Physical Chemistry B, 2017Co-Authors: Jiyong Park, Thomas A Bobik, Sunny Chun, K N Houk, Todd O YeatesAbstract:
Bacterial Microcompartments are giant protein-based organelles that encapsulate special metabolic pathways in diverse bacteria. Structural and genetic studies indicate that metabolic substrates enter these Microcompartments by passing through the central pores in hexameric assemblies of shell proteins. Limiting the escape of toxic metabolic intermediates created inside the Microcompartments would confer a selective advantage for the host organism. Here, we report the first molecular dynamics (MD) simulation studies to analyze small-molecule transport across a Microcompartment shell. PduA is a major shell protein in a Bacterial Microcompartment that metabolizes 1,2-propanediol via a toxic aldehyde intermediate, propionaldehyde. Using both metadynamics and replica-exchange umbrella sampling, we find that the pore of the PduA hexamer has a lower energy barrier for passage of the propanediol substrate compared to the toxic propionaldehyde generated within the Microcompartment. The energetic effect is consiste…
structure of a novel 13 nm dodecahedral nanocage assembled from a redesigned Bacterial Microcompartment shell proteinChemical Communications, 2016Co-Authors: Julien Jorda, M C Thompson, D J Leibly, Todd O YeatesAbstract:
We report the crystal structure of a novel 60-subunit dodecahedral cage that results from self-assembly of a re-engineered version of a natural protein (PduA) from the Pdu Microcompartment shell. Biophysical data illustrate the dependence of assembly on solution conditions, opening up new applications in Microcompartment studies and nanotechnology.
selective molecular transport through the protein shell of a Bacterial Microcompartment organelleProceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Chiranjit Chowdhury, A H Pang, Todd O Yeates, Sharmistha Sinha, Sunny Chun, Michael R. Sawaya, Thomas A BobikAbstract:
Bacterial Microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for Microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) Microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu Microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.