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Plegaria, Jefferson S.

Publications and source records attributed to Plegaria, Jefferson S..

4 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures

Bacterial Microcompartments (BMCs) are organelles composed of a selectively permeable protein shell that encapsulates enzymes involved in CO2 fixation (carboxysomes) or carbon catabolism (metabolosomes). Confinement of sequential reactions by the BMC shell presumably increases the efficiency of the pathway by reducing the crosstalk of metabolites, release of toxic intermediates, and accumulation of inhibitory products. Because BMCs are composed entirely of protein and self-assemble, they are an emerging platform for engineering nanoreactors and molecular scaffolds. However, testing designs for assembly and function through in vivo expression is labor-intensive and has limited the potential of BMCs in bioengineering. Here, we developed a new method for in vitro assembly of defined nanoscale BMC architectures: shells and nanotubes. By inserting a “protecting group”, a short ubiquitin-like modifier (SUMO) domain, self-assembly ofshell proteins in vivo was thwarted, enabling preparation of concentrates of shell building blocks. Addition of the cognate protease removes the SUMO domain and subsequent mixing of the constituent shell proteins in vitro results in the self-assembly of three types of supramolecular architectures: a metabolosome shell, a carboxysome shell, and a BMC protein-based nanotube. We next applied our method to generate a metabolosome shell engineered with a hyper-basic luminal surface, allowing for the encapsulation of biotic or abiotic cargos functionalized with an acidic accessory group. This is the first demonstration of using charge complementarity to encapsulate diverse cargos in BMC shells. Finally, our work provides a generally applicable method for in vitro assembly of natural and engineered BMC-based architectures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Structural and Functional Characterization of a Short-Chain Flavodoxin Associated with a Noncanonical 1,2-Propanediol Utilization Bacterial Microcompartment

Bacterial microcompartments (BMCs) are proteinaceous organelles that encapsulate enzymes involved in CO 2 fixation (carboxysomes). or carbon catabolism (metabolosomes). Metabolosomes share a common core of enzymes and a distinct signature enzyme for substrate degradation that defines the function of the BMC (e,g., propanediol or ethanolamine utilization BMCs, or glycyl-radical enzyme microcompartments). Loci encoding metabolosomes also typically contain genes for proteins that support organelle function, such as regulation, transport of substrate, and cofactor (e.g., vitamin B-12) synthesis and recycling. Flavoproteins are frequently among these ancillary gene products, suggesting that these redox active proteins play an undetermined function in many metabolosomes. Here in this paper, we report the first characterization of a BMC-associated flavodoxin (Fld1C), a small flavoprotein, derived from the noncanonical 1,2-propanediol utilization BMC locus (PDU1C) of Lactobacillus reuteri. The 2.0 angstrom X-ray structure of Fld1C displays the alpha/beta flavodoxin fold, which noncovalently binds a single flavin mononucleotide molecule. Fld1C is a short-chain flavodoxin with redox potentials of -240 +/- 3 mV oxidized/semiquinone and -344 +/- 1 mV semiquinone/hydroquinone versus the standard hydrogen electrode at pH 7.5. It can participate in an electron transfer reaction with a photoreductant to form a stable semiquinone species. Collectively, our structural and functional results suggest that PDU1C BMCs encapsulate Fld1C to store and transfer electrons for the reactivation and/or recycling of the B 12 cofactor utilized by the signature enzyme.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. Here, we also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.

59 BASIC BIOLOGICAL SCIENCES↗