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DOE OSTI2023

Less Is More: Oligomer Extraction and Hydrothermal Annealing Increase PDMS Adhesion Forces for Materials Studies and for Biology-Focused Microfluidic Applications

Cues in the micro-environment are key determinants in the emergence of complex cellular morphologies and functions. Primary among these is the presence of neighboring cells that form networks. For high-resolution analysis, it is crucial to develop micro-environments that permit exquisite control of network formation. This is especially true in cell science, tissue engineering, and clinical biology. We introduce a new approach for assembling polydimethylsiloxane (PDMS)-based microfluidic environments that enhances cell network formation and analyses. We report that the combined processes of PDMS solvent-extraction and hydrothermal annealing create unique conditions that produce high-strength bonds between solvent-extracted PDMS (E-PDMS) and glass—properties not associated with conventional PDMS. Extraction followed by hydrothermal annealing removes unbound oligomers, promotes polymer cross-linking, facilitates covalent bond formation with glass, and retains the highest biocompatibility. Herein, our extraction protocol accelerates oligomer removal from 5 to 2 days. Resulting microfluidic platforms are uniquely suited for cell-network studies owing to high adhesion forces, effectively corralling cellular extensions and eliminating harmful oligomers. We demonstrate the simple, simultaneous actuation of multiple microfluidic domains for invoking ATP- and glutamate-induced Ca 2+ signaling in glial-cell networks. These E-PDMS modifications and flow manipulations further enable microfluidic technologies for cell-signaling and network studies as well as novel applications.

36 MATERIALS SCIENCE↗

DOE OSTI2022

Advances in the Understanding of the Lifecycle of Photosystem II

Photosystem II is a light-driven water-plastoquinone oxidoreductase present in cyanobacteria, algae and plants. It produces molecular oxygen and protons to drive ATP synthesis, fueling life on Earth. As a multi-subunit membrane-protein-pigment complex, Photosystem II undergoes a dynamic cycle of synthesis, damage, and repair known as the Photosystem II lifecycle, to maintain a high level of photosynthetic activity at the cellular level. Cyanobacteria, oxygenic photosynthetic bacteria, are frequently used as model organisms to study oxygenic photosynthetic processes due to their ease of growth and genetic manipulation. The cyanobacterial PSII structure and function have been well-characterized, but its lifecycle is under active investigation. In this review, advances in studying the lifecycle of Photosystem II in cyanobacteria will be discussed, with a particular emphasis on new structural findings enabled by cryo-electron microscopy. These structural findings complement a rich and growing body of biochemical and molecular biology research into Photosystem II assembly and repair.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Cyclic Electron Flow-Coupled Proton Pumping in Synechocystis sp. PCC6803 Is Dependent upon NADPH Oxidation by the Soluble Isoform of Ferredoxin:NADP-Oxidoreductase

Ferredoxin:NADP-oxidoreductase (FNR) catalyzes the reversible exchange of electrons between ferredoxin (Fd) and NADP(H). Reduction of NADP+ by Fd via FNR is essential in the terminal steps of photosynthetic electron transfer, as light-activated electron flow produces NADPH for CO2 assimilation. FNR also catalyzes the reverse reaction in photosynthetic organisms, transferring electrons from NADPH to Fd, which is important in cyanobacteria for respiration and cyclic electron flow (CEF). The cyanobacterium Synechocystis sp. PCC6803 possesses two isoforms of FNR, a large form attached to the phycobilisome (FNRL) and a small form that is soluble (FNRS). While both isoforms are capable of NADPH oxidation or NADP+ reduction, FNRL is most abundant during typical growth conditions, whereas FNRS accumulates under stressful conditions that require enhanced CEF. Because CEF-driven proton pumping in the light–dark transition is due to NDH-1 complex activity and they are powered by reduced Fd, CEF-driven proton pumping and the redox state of the PQ and NADP(H) pools were investigated in mutants possessing either FNRL or FNRS. We found that the FNRS isoform facilitates proton pumping in the dark–light transition, contributing more to CEF than FNRL. FNRL is capable of providing reducing power for CEF-driven proton pumping, but only after an adaptation period to illumination. The results support that FNRS is indeed associated with increased cyclic electron flow and proton pumping, which is consistent with the idea that stress conditions create a higher demand for ATP relative to NADPH.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Photosynthetic Electron Flows and Networks of Metabolite Trafficking to Sustain Metabolism in Photosynthetic Systems

Photosynthetic eukaryotes have metabolic pathways that occur in distinct subcellular compartments. However, because metabolites synthesized in one compartment, including fixed carbon compounds and reductant generated by photosynthetic electron flows, may be integral to processes in other compartments, the cells must efficiently move metabolites among the different compartments. This review examines the various photosynthetic electron flows used to generate ATP and fixed carbon and the trafficking of metabolites in the green alga Chlamydomomas reinhardtii; information on other algae and plants is provided to add depth and nuance to the discussion. We emphasized the trafficking of metabolites across the envelope membranes of the two energy powerhouse organelles of the cell, the chloroplast and mitochondrion, the nature and roles of the major mobile metabolites that move among these compartments, and the specific or presumed transporters involved in that trafficking. These transporters include sugar-phosphate (sugar-P)/inorganic phosphate (Pi) transporters and dicarboxylate transporters, although, in many cases, we know little about the substrate specificities of these transporters, how their activities are regulated/coordinated, compensatory responses among transporters when specific transporters are compromised, associations between transporters and other cellular proteins, and the possibilities for forming specific ‘megacomplexes’ involving interactions between enzymes of central metabolism with specific transport proteins. Finally, we discuss metabolite trafficking associated with specific biological processes that occur under various environmental conditions to help to maintain the cell’s fitness. These processes include C4 metabolism in plants and the carbon concentrating mechanism, photorespiration, and fermentation metabolism in algae.

Plant Sciences↗

DOE OSTI2020

Flexibility in the Energy Balancing Network of Photosynthesis Enables Safe Operation under Changing Environmental Conditions

Given their ability to harness chemical energy from the sun and generate the organic compounds necessary for life, photosynthetic organisms have the unique capacity to act simultaneously as their own power and manufacturing plant. This dual capacity presents many unique challenges, chiefly that energy supply must be perfectly balanced with energy demand to prevent photodamage and allow for optimal growth. From this perspective, we discuss the energy balancing network using recent studies and a quantitative framework for calculating metabolic ATP and NAD(P)H demand using measured leaf gas exchange and assumptions of metabolic demand. We focus on exploring how the energy balancing network itself is structured to allow safe and flexible energy supply. We discuss when the energy balancing network appears to operate optimally and when it favors high capacity instead. We also present the hypothesis that the energy balancing network itself can adapt over longer time scales to a given metabolic demand and how metabolism itself may participate in this energy balancing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Advances in Genes-Encoding Transporters for Cadmium Uptake, Translocation, and Accumulation in Plants

Cadmium (Cd) is a heavy metal that is highly toxic for plants, animals, and human beings. A better understanding of the mechanisms involved in Cd accumulation in plants is beneficial for developing strategies for either the remediation of Cd-polluted soils using hyperaccumulator plants or preventing excess Cd accumulation in the edible parts of crops and vegetables. As a ubiquitous heavy metal, the transport of Cd in plant cells is suggested to be mediated by transporters for essential elements such as Ca, Zn, K, and Mn. Identification of the genes encoding Cd transporters is important for understanding the mechanisms underlying Cd uptake, translocation, and accumulation in either crop or hyperaccumulator plants. Recent studies have shown that the transporters that mediate the uptake, transport, and accumulation of Cd in plants mainly include members of the natural resistance-associated macrophage protein (Nramp), heavy metal-transporting ATPase (HMA), zinc and iron regulated transporter protein (ZIP), ATP-binding cassette (ABC), and yellow stripe-like (YSL) families. Here, we review the latest advances in the research of these Cd transporters and lay the foundation for a systematic understanding underlying the molecular mechanisms of Cd uptake, transport, and accumulation in plants.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Simplification of the Grid Model and its Impact on the Analysis of Electrical Power Systems

Here, this paper analyzes the impact of the use of Kron reduction on the state variables of a three-phase electrical system, even when it does not meet the necessary conditions for its application. Reduction is applied to a power line model to eliminate the equation corresponding to the neutral conductor of the line. The ATP program is used to model and simulate the behavior of an electrical system considering different degrees of disequilibrium as a reference for the comparison of results. The results show that under certain conditions of disequilibrium the Kron reduction can lead to significant errors in the state variables of the system.

Electric Power Systems↗

DOE OSTI2026

The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex

ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.

Zhang, Bo [Department of Chemical Engineering, Uni↗

DOE OSTI2022

Extracellular electron transfer increases fermentation in lactic acid bacteria via a hybrid metabolism

Energy conservation in microorganisms is classically categorized into respiration and fermentation; however, recent work shows some species can use mixed or alternative bioenergetic strategies. We explored the use of extracellular electron transfer for energy conservation in diverse lactic acid bacteria (LAB), microorganisms that mainly rely on fermentative metabolism and are important in food fermentations. The LAB Lactiplantibacillus plantarum uses extracellular electron transfer to increase its NAD + /NADH ratio, generate more ATP through substrate-level phosphorylation, and accumulate biomass more rapidly. This novel, hybrid metabolism is dependent on a type-II NADH dehydrogenase (Ndh2) and conditionally requires a flavin-binding extracellular lipoprotein (PplA) under laboratory conditions. It confers increased fermentation product yield, metabolic flux, and environmental acidification in laboratory media and during kale juice fermentation. The discovery of a single pathway that simultaneously blends features of fermentation and respiration in a primarily fermentative microorganism expands our knowledge of energy conservation and provides immediate biotechnology applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a ‘pincer-like’ conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5–nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

The relationship between gene traits and transcription in soil microbial communities varies by environmental stimulus

Codon and nucleotide frequencies are known to relate to the rate of gene transcription, yet how these traits shape transcriptional profiles of soil microbial communities remains unclear. Here we test the prediction that functional genes with high codon optimization and energetically lower cost nucleotides (i.e., nucleotides requiring less adenosine triphosphate (ATP) for synthesis) have higher transcriptional expression in a soil microbial community. In laboratory incubations, we subjected an agricultural soil to two separate short-term environmental changes: labile carbon (glucose) addition or a sudden 30-min increase in temperature from 20 °C to 60 °C. Using the total genomic codon frequencies to predict preferred codon usage for each taxon, we then estimated codon optimization for each transcript. On the community level, we found a higher average level of codon optimization after the addition of glucose. Synonymous nucleotide composition in the transcript pool also shifted towards energetically cheaper nucleotides, favoring uracil (U) over adenine (A) and cytosine (C) over guanine (G). Similarly, we found that encoded amino acid usage shifted towards energetically cheaper amino acids in response to labile carbon. In contrast, in communities responding to heat shock, there were no significant differences in the averaged gene traits of expressed transcripts. We used metagenome-assembled-genomes to further examine the ability of gene traits to predict transcriptional responses within and between taxa. We found that traits of individual genes could not reliably predict the level of transcription of a gene within or between taxa—highlighting the limits of this approach. However, we did find that when traits were averaged across several related genes, codon optimization was able to predict levels of transcription in metabolic pathways associated with growth and nutrient uptake in response to glucose. Similar relationships were not observed in response to heat, or for functions associated with stress—such as genes associated with sporulation or heat shock. These results demonstrate that gene traits, such as codon usage, nucleotide selection, and amino acid selection, relate to the transcriptional expression of genes in soil microbial communities and suggests that these relationships may be dependent on both gene function and the specific type of environmental stimuli.

Biological and medical sciences↗

DOE OSTI2021

BETO 2021 Peer Review - Cyanobacteria Photosynthetic Energy Platform (1.3.4.301)

Cyanobacteria Photosynthetic Energy Platform (WBS 1.3.4.301) The primary challenge in algal technology is low energy conversion efficiency, currently up to a few percentages from photon energy to biomass energy. Most incoming solar energy is lost in the processes of photosynthesis, through mechanisms that are not fully understood. There is large room for improvement. This project develops cyanobacteria genetic tools to improve photosynthetic efficiency and biomass productivity through manipulation of energy regulation mechanisms. Increasing algal biomass productivity by 20% translates to $2 reduction of MFSP. Genetic manipulation is an essential tool in understanding and improving algal productivity. While genetic manipulation is challenging for most algae, model cyanobacteria, with in-house mutant library and genetic engineering toolboxes, allow for rapid hypothesis testing and transfer of lessons to other cyanobacteria and eukaryotic algae. We developed Energome (energy-ome) concept to guide novel engineering strategies and unleash unused potential in photosynthesis. Energome consists of cellular energy management mechanisms. For example, we found that cyanobacteria use futile cycles around glycogen and sucrose to dissipate ATP. By genetic modification, the model cyanobacterium Synechocystis 6803 showed higher energy levels, increased photosynthetic capacity, and 20% improvement in biomass yield in simulated outdoor light conditions.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

DOE OSTI2022

Compositions and methods for inhibiting kinase activity

The present invention features therapeutic compositions comprising an agent that specifically binds to a PIF pocket of Aurora A kinase and an agent that specifically binds to an ATP-binding site of Aurora A kinase, and the use of the therapeutic compositions to modulate Aurora A kinase for the treatment of cancer.

Kern, Douglas↗

DOE OSTI2020

Reveal of Uranium Bioremediation Mechanisms by Bacillus Species through Proteomics Studies

Radionuclides, such as Uranium (U) and heavy metals continue to pose threats to the ecosystem health and processes at the Department of Energy (DOE) managed, Savannah River Site (SRS), located along the Savannah River near Aiken, SC. Such co-contaminated environments are difficult to remediate using conventional excavation and disposal or pump-and-treat approaches. Globally, more than 109 tons of uranium contaminated areas pose a long term threat to human and ecological health. Even with presented low concentrations in the brain, central nervous effects are still observed. Uranium and depleted uranium (DU) have long term effects on the kidneys. Some small general health effects include severe headaches and breathing problems. Uranus ions are toxic to living cells because they inhibit metabolism of carbohydrates by blocking ATP binding sites. Bioremediation by microorganisms represents an alternative solution, which is advantageous because of the possibility of biosorbent regeneration, cost-effectiveness, increased metal removal and easy recovery of some valuable metals. Bacillus sp. bacterium was previously used in the bioremediation of heavy metals in coal mine run off waters of SRS. However, its ability to bioremediate uranium was unknown so far. Hence, in the present study, uranium bioremediation by Bacillus sp. bacterium was investigated. The mechanism of bioremediation was also revealed through proteomics studies. Heavy metals contamination poses a serious threat to water, soil and human health. Soil and water are contaminated due to excessive exploitation of uranium mines for generation of nuclear energy and weaponry. It is not degradable easily and persist in soil and water for a long period of time due to its long half- life. Savannah river site (SRS) is one of the uranium contaminated sites. The physical or chemical remediation techniques are costly and complex. Microbial system approaches with competent bacteria has received increased attention due to its adaptability in various environmental matrices and cost effectiveness. However, even though there are multiple suggested pathways (F1), the specific mechanisms that drive this behavior are still unclear, especially with popular microorganisms such as Bacillus species. In a previous research (Ibeanusi et al, 2003) Bacillus sp. was shown to detoxify and precipitate a variety of heavy metals in coal pile runoff waters of SRS site. Additionally, genomic analysis demonstrated that this microorganism posses multiple attributes for chemical transport regulation and metabolic pathways. U remediation occurs during the first 20 hours of exposure. During this time period, Bacillus sp. may work primarily under two mechanisms - sorption and accumulation. These two mechanisms simultaneously work to protect the microorganism from high concentrations. Figure 10 demonstrates that certain proteins are up regulated and down regulated under extreme stressful conditions. Membrane fraction proteins were up regulated. Cytosolic fraction proteins were down regulated. This protein information coincides with the adsorption behavior. Bacillus sp. is a good candidate for U remediation at various concentrations.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2023

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT)

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04

Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments - high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO 2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Altogether, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequencebased methods.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Heat activates the AAA+ HslUV protease by melting an axial autoinhibitory plug

At low temperatures, protein degradation by the AAA+ HslUV protease is very slow. New crystal structures reveal that residues in the intermediate domain of the HslU 6 unfoldase can plug its axial channel, blocking productive substrate binding and subsequent unfolding, translocation, and degradation by the HslV 12 peptidase. Biochemical experiments with wild-type and mutant enzymes support a model in which heat-induced melting of this autoinhibitory plug activates HslUV proteolysis.

59 BASIC BIOLOGICAL SCIENCES↗