The translocation activity of Rad54 reduces crossover outcomes during homologous recombination.

Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that requires the selection of an appropriate DNA sequence to facilitate repair. Selection occurs during a homology search that must be executed rapidly and with high fidelity. Failure to efficiently perform the homology search can result in complex intermediates that generate genomic rearrangements, a hallmark of human cancers. Rad54 is an ATP dependent DNA motor protein that functions during the homology search by regulating the recombinase Rad51. How this regulation reduces genomic exchanges is currently unknown. To better understand how Rad54 can reduce these outcomes, we evaluated several amino acid mutations in Rad54 that were identified in the COSMIC database. COSMIC is a collection of amino acid mutations identified in human cancers. These substitutions led to reduced Rad54 function and the discovery of a conserved motif in Rad54. Through genetic, biochemical and single-molecule approaches, we show that disruption of this motif leads to failure in stabilizing early strand invasion intermediates, causing increased crossovers between homologous chromosomes. Our study also suggests that the translocation rate of Rad54 is a determinant in balancing genetic exchange. The latch domain's conservation implies an interaction likely fundamental to eukaryotic biology.

Rad53 regulates the lifetime of Rdh54 at homologous recombination intermediates.

Rdh54 is a conserved DNA translocase that participates in homologous recombination (HR), DNA checkpoint adaptation, and chromosome segregation. Saccharomyces cerevisiae Rdh54 is a known target of the Mec1/Rad53 signaling axis, which globally protects genome integrity during DNA metabolism. While phosphorylation of DNA repair proteins by Mec1/Rad53 is critical for HR progression little is known about how specific post translational modifications alter HR reactions. Phosphorylation of Rdh54 is linked to protection of genomic integrity but the consequences of modification remain poorly understood. Here, we demonstrate that phosphorylation of the Rdh54 C-terminus by the effector kinase Rad53 regulates Rdh54 clustering activity as revealed by single molecule imaging. This stems from phosphorylation dependent and independent interactions between Rdh54 and Rad53. Genetic assays reveal that loss of phosphorylation leads to phenotypic changes resulting in loss-of-heterozygosity (LOH) outcomes. Our data highlight Rad53 as a key regulator of HR intermediates through activation and attenuation of Rdh54 motor function.

Rdh54 stabilizes Rad51 at displacement loop intermediates to regulate genetic exchange between chromosomes.

Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair damaged DNA, HR uses an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulation of DNA sequence transfer is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH), and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR. Several existing phenotypes in rdh54Δ strains suggest that Rdh54 may regulate effective exchange of DNA during HR. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information during DNA repair. Our data indicate that RDH54 regulates DNA strand exchange by stabilizing Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 acts in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, favors non-crossover outcomes. From these results, we propose a model for how Rdh54 may kinetically regulate strand exchange during homologous recombination.

Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate.

Formate has great potential to function as a feedstock for biorefineries because it can be sustainably produced by a variety of processes that don't compete with agricultural production. However, naturally formatotrophic organisms are unsuitable for large-scale cultivation, difficult to engineer, or have inefficient native formate assimilation pathways. Thus, metabolic engineering needs to be developed for model industrial organisms to enable efficient formatotrophic growth. Here, we build a prototype synthetic formate utilizing bacterial microcompartment (sFUT) encapsulating the oxygen-sensitive glycyl radical enzyme pyruvate formate lyase and a phosphate acyltransferase to convert formate and acetyl-phosphate into the central biosynthetic intermediate pyruvate. This metabolic module offers a defined environment with a private cofactor coenzyme A that can cycle efficiently between the encapsulated enzymes. To facilitate initial design-build-test-refine cycles to construct an active metabolic core, we used a "wiffleball" architecture, defined as an icosahedral bacterial microcompartment (BMC) shell with unoccupied pentameric vertices to freely permit substrate and product exchange. The resulting sFUT prototype wiffleball is an active multi enzyme synthetic BMC functioning as platform technology.

Electronic structure and energetics of a heterodimeric BChl g'/Chl a' special pair generated by exposure of Heliomicrobium modesticaldum to dioxygen.

Heliobacteria are anoxygenic phototrophs that have a Type I homodimeric reaction center containing bacteriochlorophyll g (BChl g). Previous experimental studies have shown that in the presence of light and dioxygen, BChl g is converted into 81-OH-chlorophyll aF (hereafter Chl aF), with an accompanying loss of light-driven charge separation. These studies suggest that the reaction center only loses the ability to transfer electrons once both BChl g' molecules of the P800 special pair have been converted to Chl aF'. The present work confirms that the partially converted BChl g'/Chl aF' special pair remains functional in samples exposed to dioxygen by demonstrating its presence using hyperfine couplings obtained from Q-band 1H ENDOR, 2D 14N HYSCORE and DFT methods. The DFT calculations of the BChl g'/BChl g' homodimeric primary donor, which are based on the recently published X-ray crystal structure, predict that the unpaired electron spin is equally delocalized over both BChl g' molecules and provide an excellent match to the experimental hyperfine couplings of the anaerobic samples. Exposure to dioxygen leads to substantial changes in the hyperfine interactions, indicative of greater localization of the unpaired electron spin. The measured hyperfine couplings are reproduced in the DFT calculations by replacing one of the BChl g' molecules of the primary donor with a Chl aF' molecule. The calculations reveal that the spin density becomes localized on BChl g' in the heterodimeric primary donor. Time-dependent DFT calculations demonstrate that conversion of either or both of the accessory BChl g molecules and/or one of the BChl g' molecules of P800 to Chl aF' results in minor effects on the energy of the charge-separated states. In contrast, if both of the BChl g' molecules of P800 are converted a large increase in the energy of the charge-separated state occurs. This suggests that the reaction center remains functional when only one half of the dimer is converted, however, conversion of both halves of the P800 dimer leads to loss of function.

Differential sensitivity to oxygen among the bacteriochlorophylls g in the type-I reaction centers of Heliobacterium modesticaldum.

The type-I, homodimeric photosynthetic reaction center (RC) of Heliobacteria (HbRC) is the only known RC in which bacteriochlorophyll g (BChl g) is found. It is also simpler than other RCs, having the smallest number of protein subunits and bound chromophores of any type-I RC. In the presence of oxygen, BChl g isomerizes to 81-hydroxychlorophyll aF (Chl aF). This naturally occurring process provides a way of altering the chlorophylls and studying the effect of these changes on energy and electron transfer. Transient absorbance difference spectroscopy reveals that triplet-state formation occurs in the antenna chlorophylls of HbRCs but does not provide site-specific information. Here, we report on an extended optically detected magnetic resonance (ODMR) study of the antenna triplet states in HbRCs with differing levels of conversion of BChl g to Chl aF. The data reveal pools of BChl g molecules with different triplet zero-field splitting parameters and different susceptibilities to chemical oxidation. By relating the detailed spectroscopic characteristics derived from the ODMR data to the recently solved crystallographic structure, we have tentatively identified BChl g molecules in which the probability of triplet formation is high and sites at which BChl g conversion is more likely, providing useful information about the fate of the excitation in the complex.

A designed bacterial microcompartment shell with tunable composition and precision cargo loading.

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.

Reaction centers of the thermophilic microaerophile, Chloracidobacterium thermophilum (Acidobacteria) I: biochemical and biophysical characterization.

Chloracidobacterium thermophilum is a microaerophilic, anoxygenic member of the green chlorophototrophic bacteria. This bacterium is the first characterized oxygen-requiring chlorophototroph with chlorosomes, the FMO protein, and homodimeric type-1 reaction centers (RCs). The RCs of C. thermophilum are also unique because they contain three types of chlorophylls, bacteriochlorophyll aP esterified with phytol, Chl aPD esterified with Δ2,6-phytadienol, and Zn-BChl aP' esterified with phytol, in the approximate molar ratio 32:24:4. The light-induced difference spectrum of these RCs had a bleaching maximum at 839 nm and also revealed an electrochromic bandshift that is probably derived from a BChl a molecule near P840+. The FX [4Fe-4S] cluster had a midpoint potential of ca. - 581 mV, and the spectroscopic properties of the P+ F X - spin-polarized radical pair were very similar to those of reaction centers of heliobacteria and green sulfur bacteria. The data further indicate that electron transfer occurs directly from A0- to FX, as occurs in other homodimeric type-1 RCs. Washing experiments with isolated membranes suggested that the PscB subunit of these reaction centers is more tightly bound than PshB in heliobacteria. Thus, the reaction centers of C. thermophilum have some properties that resemble other homodimeric reaction centers but also have specific properties that are more similar to those of Photosystem I. These differences probably contribute to protection of the electron transfer chain from oxygen, contributing to the oxygen tolerance of this microaerophile.

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 of shell 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. Collectively, our work provides a generally applicable method for in vitro assembly of natural and engineered BMC-based architectures.

Light-Driven Chloride Transport Kinetics of Halorhodopsin.

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl- transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl-/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.

BciD Is a Radical S-Adenosyl-l-methionine (SAM) Enzyme That Completes Bacteriochlorophyllide e Biosynthesis by Oxidizing a Methyl Group into a Formyl Group at C-7.

Green bacteria are chlorophotorophs that synthesize bacteriochlorophyll (BChl) c, d, or e, which assemble into supramolecular, nanotubular structures in large light-harvesting structures called chlorosomes. The biosynthetic pathways of these chlorophylls are known except for one reaction. Null mutants of bciD, which encodes a putative radical S-adenosyl-l-methionine (SAM) protein, are unable to synthesize BChl e but accumulate BChl c; however, it is unknown whether BciD is sufficient to convert BChl c (or its precursor, bacteriochlorophyllide (BChlide) c) into BChl e (or BChlide e). To determine the function of BciD, we expressed the bciD gene of Chlorobaculum limnaeum strain DSMZ 1677T in Escherichia coli and purified the enzyme under anoxic conditions. Electron paramagnetic resonance spectroscopy of BciD indicated that it contains a single [4Fe-4S] cluster. In assays containing SAM, BChlide c or d, and sodium dithionite, BciD catalyzed the conversion of SAM into 5'-deoxyadenosine and BChlide c or d into BChlide e or f, respectively. Our analyses also identified intermediates that are proposed to be 71-OH-BChlide c and d Thus, BciD is a radical SAM enzyme that converts the methyl group of BChlide c or d into the formyl group of BChlide e or f This probably occurs by a mechanism involving consecutive hydroxylation reactions of the C-7 methyl group to form a geminal diol intermediate, which spontaneously dehydrates to produce the final products, BChlide e or BChlide f The demonstration that BciD is sufficient to catalyze the conversion of BChlide c into BChlide e completes the biosynthetic pathways for all "Chlorobium chlorophylls."

Presence of a [3Fe-4S] cluster in a PsaC variant as a functional component of the photosystem I electron transfer chain in Synechococcus sp. PCC 7002.

A site-directed C14G mutation was introduced into the stromal PsaC subunit of Synechococcus sp. strain PCC 7002 in vivo in order to introduce an exchangeable coordination site into the terminal FB [4Fe-4S] cluster of Photosystem I (PSI). Using an engineered PSI-less strain (psaAB deletion), psaC was deleted and replaced with recombinant versions controlled by a strong promoter, and the psaAB deletion was complemented. Modified PSI accumulated at lower levels in this strain and supported slower photoautotrophic growth than wild type. As-isolated PSI complexes containing PsaCC14G showed resonances with g values of 2.038 and 2.007 characteristic of a [3Fe-4S]1+ cluster. When the PSI complexes were illuminated at 15 K, these resonances partially disappeared and two new sets of resonances appeared. The majority set had g values of 2.05, 1.95, and 1.85, characteristic of FA-, and the minority set had g values of 2.11, 1.90, and 1.88 from FB' in the modified site. The S = 1/2 spin state of the latter implied the presence of a thiolate as the terminal ligand. The [3Fe-4S] clusters could be partially reconstituted with iron, producing a larger population of [4Fe-4S] clusters. Rates of flavodoxin reduction were identical in PSI complexes isolated from wild type and the PsaCC14G variant strain; this implied equivalent capacity for forward electron transfer in PSI complexes that contained [3Fe-4S] and [4Fe-4S] clusters. The development of this cyanobacterial strain is a first step toward translation of in vitro PSI-based biosolar molecular wire systems in vivo and provides new insights into the formation of Fe/S clusters.

15N photo-CIDNP MAS NMR analysis of reaction centers of Chloracidobacterium thermophilum.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by 15N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a' (Zn-BChl a') (Tsukatani et al. in J Biol Chem 287:5720-5732, 2012). Based upon experimental and quantum chemical 15N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a'. Chl a and 81-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.

Improving extraction and post-purification concentration of membrane proteins.

Membrane proteins (MPs), despite being critically important drug targets for the pharmaceutical industry, are difficult to study due to challenges in obtaining high yields of functional protein. Most current extraction efforts use specialized non-ionic detergents to solubilize and stabilize MPs, with MPs being concentrated by ultrafiltration (UF). However, many detergents are retained during the UF step, which can destabilize MPs and/or interfere with their characterization. Here, we studied the influence of detergent selection on the extraction and UF-based concentration of biomedically-relevant MPs, the light-driven sodium and chloride transporters, KR2 and halorhodopsin (pHR) which are also model proteins for more complex mammalian rhodopsins. We also designed a flat-bottomed centrifugal filter that can concentrate MPs with enhanced removal of free detergents by promoting concentration polarization (CP). We tested the performance of this new filter using four commonly employed MP detergents, octyl-ß-D maltoside (OM), decyl-ß-D maltoside (DM), dodecyl-ß-D maltoside (DDM) and octyl-ß-D glucoside (OG), over a range of detergent and salt concentrations. Detergent passage is significantly higher for the flat-bottomed filter achieving up to 2-fold greater sieving of detergent in DM-solubilized pHR system due to the high degree of CP. We observe more efficient, up to 5-fold higher extraction of KR2 in the presence of a longer 12-carbon alkyl chain detergent, DDM compared to a shorter 8-carbon detergent, OM. Assuming complete binding and elution of the extracted protein, DDM-based extraction of KR2 could lead to a potential 7-fold improvement in purification yields compared to conventional methods which yield ∼1 mg MP per liter of cell culture. However, the longer chain detergents like DDM form larger micelles that are difficult to remove by UF. Thus, there exists a trade-off between choosing a detergent that will enable efficient extraction of MP while showing easier removal during subsequent UF. The extraction efficiency and UF-based separation of detergent micelles provide insights for other applications involving detergent-mediated separation/extraction.

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 CO2 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 B12) 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, 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 Å X-ray structure of Fld1C displays the α/ß 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 B12 cofactor utilized by the signature enzyme.

Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor.

Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn2+-inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn2+ uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA7942) and its cognate SmtB7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA6803) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster ß-carotene 15,15'-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002. IMPORTANCE: This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn2+-inducible system, which is based on the smtA7942 operator/promoter and smtB7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

Thermodynamics of the Electron Acceptors in Heliobacterium modesticaldum: An Exemplar of an Early Homodimeric Type I Photosynthetic Reaction Center.

The homodimeric type I reaction center in heliobacteria is arguably the simplest known pigment-protein complex capable of conducting (bacterio)chlorophyll-based conversion of light into chemical energy. Despite its structural simplicity, the thermodynamics of the electron transfer cofactors on the acceptor side have not been fully investigated. In this work, we measured the midpoint potential of the terminal [4Fe-4S](2+/1+) cluster (FX) in reaction centers from Heliobacterium modesticaldum. The FX cluster was titrated chemically and monitored by (i) the decrease in the level of stable P800 photobleaching by optical spectroscopy, (ii) the loss of the light-induced g ≈ 2 radical from P800(+•) following a single-turnover flash, (iii) the increase in the low-field resonance at 140 mT attributed to the S = (3)/2 ground spin state of FX(-), and (iv) the loss of the spin-correlated P800(+) FX(-) radical pair following a single-turnover flash. These four techniques led to similar estimations of the midpoint potential for FX of -502 ± 3 mV (n = 0.99), -496 ± 2 mV (n = 0.99), -517 ± 10 mV (n = 0.65), and -501 ± 4 mV (n = 0.84), respectively, with a consensus value of -504 ± 10 mV (converging to n = 1). Under conditions in which FX is reduced, the long-lived (∼15 ms) P800(+) FX(-) state is replaced by a rapidly recombining (∼15 ns) P800(+)A0(-) state, as shown by ultrafast optical experiments. There was no evidence of the presence of a P800(+) A1(-) spin-correlated radical pair by electron paramagnetic resonance (EPR) under these conditions. The midpoint potentials of the two [4Fe-4S](2+/1+) clusters in the low-molecular mass ferredoxins were found to be -480 ± 11 mV/-524 ± 13 mV for PshBI, -453 ± 6 mV/-527 ± 6 mV for PshBII, and -452 ± 5 mV/-533 ± 8 mV for HM1_2505 as determined by EPR spectroscopy. FX is therefore suitably poised to reduce one [4Fe-4S](2+/1+) cluster in these mobile electron carriers. Using the measured midpoint potential of FX and a quasi-equilibrium model of charge recombination, the midpoint potential of A0 was estimated to be -854 mV at room temperature. The midpoint potentials of A0 and FX are therefore 150-200 mV less reducing than their respective counterparts in Photosystem I of cyanobacteria and plants. This places the redox potential of the FX cluster in heliobacteria approximately equipotential to the highest-potential iron-sulfur cluster (FA) in Photosystem I, consistent with its assignment as the terminal electron acceptor.

Concentrating membrane proteins using ultrafiltration without concentrating detergents.

Membrane proteins (MPs) are of rapidly growing interest in the design of pharmaceutical products, novel sensors, and synthetic membranes. Ultrafiltration (UF) using commercially available centrifugal concentrators is typically employed for laboratory-scale concentration of low-yield MPs, but its use is accompanied by a concomitant increase in concentration of detergent micelles. We present a detailed analysis of the hydrodynamic processes that control detergent passage during ultrafiltration of MPs and propose methods to optimize detergent passage during protein concentration in larger-scale membrane processes. Experiments were conducted using nonionic detergents, octyl-ß-D glucoside (OG), and decyl-ß-D maltoside (DM) with the bacterial water channel protein, Aquaporin Z (AqpZ) and the light driven chloride pump, halorhodopsin (HR), respectively. The observed sieving coefficient (So ), a measure of detergent passage, was evaluated in both stirred cell and centrifugal systems. So for DM and OG increased with increasing filtrate flux and decreasing shear rates in the stirred cell, that is, with increasing concentration polarization (CP). Similar effects were observed during filtration of MP-detergent (MPD) micelles. However, lower transmission was observed in the centrifugal system for both detergent and MPD systems. This is attributed to free convection-induced shear and hence reduced CP along the membrane surface during centrifugal UF. Thus to concentrate MPs without retention of detergent, design of UF systems that promote CP is required. Biotechnol. Bioeng. 2016;113: 2122-2130. © 2016 Wiley Periodicals, Inc.

Electrochemical cofactor recycling of bacterial microcompartments.

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B12) reductase and, curiously, found it in many unrelated BMC types that do not employ B12 cofactors. We propose NAD+ regeneration as a new function of this enzyme and name it MNdh, for Metabolosome NADH dehydrogenase. Its partner shell protein BMC-TSE assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, EPR spectroscopy, protein voltammetry and structural modeling verified with X-ray footprinting. This discovery represents a new paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

Heterologous Assembly of Pleomorphic Bacterial Microcompartment Shell Architectures Spanning the Nano- to Microscale.

Many bacteria use protein-based organelles known as bacterial microcompartments (BMCs) to organize and sequester sequential enzymatic reactions. Regardless of their specialized metabolic function, all BMCs are delimited by a shell made of multiple structurally redundant, yet functionally diverse, hexameric (BMC-H), pseudohexameric/trimeric (BMC-T), or pentameric (BMC-P) shell protein paralogs. When expressed without their native cargo, shell proteins have been shown to self-assemble into 2D sheets, open-ended nanotubes, and closed shells of ≈40 nm diameter that are being developed as scaffolds and nanocontainers for applications in biotechnology. Here, by leveraging a strategy for affinity-based purification, it is demonstrated that a wide range of empty synthetic shells, many differing in end-cap structures, can be derived from a glycyl radical enzyme-associated microcompartment. The range of pleomorphic shells observed, which span ≈2 orders of magnitude in size from ≈25 nm to ≈1.8 µm, reveal the remarkable plasticity of BMC-based biomaterials. In addition, new capped nanotube and nanocone morphologies are observed that are consistent with a multicomponent geometric model in which architectural principles are shared among asymmetric carbon, viral protein, and BMC-based structures.