Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “cyanobacteria”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Evolutionary diversity of proton and water channels on the oxidizing side of photosystem II and their relevance to function

One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. Here, this process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn 4 CaO 5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the ‘water wheel’ to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624–12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4 channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn 4 CaO 5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S 0 → S 1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421–425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624–12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831–15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural comparison of allophycocyanin variants reveals the molecular basis for their spectral differences

Abstract Allophycocyanins are phycobiliproteins that absorb red light and transfer the energy to the reaction centers of oxygenic photosynthesis in cyanobacteria and red algae. Recently, it was shown that some allophycocyanins absorb far-red light and that one subset of these allophycocyanins, comprising subunits from the ApcD4 and ApcB3 subfamilies (FRL-AP), form helical nanotubes. The lowest energy absorbance maximum of the oligomeric ApcD4-ApcB3 complexes occurs at 709 nm, which is unlike allophycocyanin (AP; ApcA-ApcB) and allophycocyanin B (AP-B; ApcD-ApcB) trimers that absorb maximally at ~ 650 nm and ~ 670 nm, respectively. The molecular bases of the different spectra of AP variants are presently unclear. To address this, we structurally compared FRL-AP with AP and AP-B, performed spectroscopic analyses on FRL-AP, and leveraged computational approaches. We show that among AP variants, the α-subunit constrains pyrrole ring A of its phycocyanobilin chromophore to different extents, and the coplanarity of ring A with rings B and C sets a baseline for the absorbance maximum of the chromophore. Upon oligomerization, the α-chromophores of all AP variants exhibit a red shift of the absorbance maximum of ~ 25 to 30 nm and band narrowing. We exclude excitonic coupling in FRL-AP as the basis for this red shift and extend the results to discuss AP and AP-B. Instead, we attribute these spectral changes to a conformational alteration of pyrrole ring D, which becomes more coplanar with rings B and C upon oligomerization. This study expands the molecular understanding of light-harvesting attributes of phycobiliproteins and will aid in designing phycobiliproteins for biotechnological applications.

Gisriel, Christopher J.↗
DOE OSTI2022

Protein–chromophore interactions controlling photoisomerization in red/green cyanobacteriochromes

Abstract Photoreceptors in the phytochrome superfamily use 15,16-photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert between two states with distinct spectral and biochemical properties. Canonical phytochromes include master regulators of plant growth and development in which light signals trigger interconversion between a red-absorbing 15Z dark-adapted state and a metastable, far-red-absorbing 15E photoproduct state. Distantly related cyanobacteriochromes (CBCRs) carry out a diverse range of photoregulatory functions in cyanobacteria and exhibit considerable spectral diversity. One widespread CBCR subfamily typically exhibits a red-absorbing 15Z dark-adapted state similar to that of phytochrome that gives rise to a distinct green-absorbing 15E photoproduct. This red/green CBCR subfamily also includes red-inactive examples that fail to undergo photoconversion, providing an opportunity to study protein–chromophore interactions that either promote photoisomerization or block it. In this work, we identified a conserved lineage of red-inactive CBCRs. This enabled us to identify three substitutions sufficient to block photoisomerization in photoactive red/green CBCRs. The resulting red-inactive variants faithfully replicated the fluorescence and circular dichroism properties of naturally occurring examples. Converse substitutions restored photoconversion in naturally red-inactive CBCRs. This work thus identifies protein–chromophore interactions that control the fate of the excited-state population in red/green cyanobacteriochromes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Mass spectrometry and spectroscopic characterization of a tetrameric photosystem I supercomplex from Leptolyngbya ohadii , a desiccation-tolerant cyanobacterium

Cyanobacteria inhabiting desert biological soil crusts face the harsh conditions of the desert. They evolved a suite of strategies toward desiccation-hydration cycles mixed with high light irradiations, etc. In this study we purified and characterized the structure and function of Photosystem I (PSI) from Leptolyngbya ohadii, a desiccation-tolerant desert cyanobacterium. We discovered that PSI forms tetrameric (PSI-Tet) aggregate. We investigated it by using sucrose density gradient centrifugation, clear native PAGE, high performance liquid chromatography, mass spectrometry (MS), time-resolved fluorescence (TRF) and time-resolved transient absorption (TA) spectroscopy. MS analysis identified the presence of two PsaB and two PsaL proteins in PSI-Tet and uniquely revealed that PsaLs are N-terminally acetylated in contrast to non-modified PsaL in the trimeric PSI from Synechocystis sp. PCC 6803. Chlorophyll (Chl) a fluorescence decay profiles of the PSI-Tet performed at 77 K revealed two emission bands at ∼690 nm and 725 nm with the former appearing only at early delay time. The main fluorescence emission peak, associated with emission from the low energy Chls a, decays within a few nanoseconds. TA studies demonstrated that the 725 nm emission band is associated with low energy Chls a with absorption band clearly resolved at ∼710 nm at 77 K. As a result, our work suggests that the heterogenous composition of PsaBs and PsaL in PSI-Tet is related with the adaptation mechanisms needed to cope with stressful conditions under which this bacterium naturally grows.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Solar water splitting Pt-nanoparticle photosystem I thylakoid systems: Catalyst identification, location and oligomeric structure

In this study, photosynthetic conversion of light energy into chemical energy occurs in sheet-like membrane-bound compartments called thylakoids and is mediated by large integral membrane protein-pigment complexes called reaction centers (RCs). Oxygenic photosynthesis of higher plants, cyanobacteria and algae requires the symbiotic linking of two RCs, photosystem II (PSII) and photosystem I (PSI), to split water and assimilate carbon dioxide. Worldwide there is a large research investment in developing RC-based hybrids that utilize the highly evolved solar energy conversion capabilities of RCs to power catalytic reactions for solar fuel generation. Of particular interest is the solar-powered production of H 2 , a clean and renewable energy source that can replace carbonbased fossil fuels and help provide for ever-increasing global energy demands. Recently, we developed thylakoid membrane hybrids with abiotic catalysts and demonstrated that photosynthetic Z-scheme electron flow from the light-driven water oxidation at PSII can drive H 2 production from PSI. One of these hybrid systems was created by self-assembling Pt-nanoparticles (PtNPs) with the stromal subunits of PSI that extend beyond the membrane plane in both spinach and cyanobacterial thylakoids. Using PtNPs as site-specific probe molecules, we report the electron microscopic (EM) imaging of oligomeric structure, location and organization of PSI in thylakoid membranes and provide the first direct visualization of photosynthetic Z-scheme solar water-splitting biohybrids for clean H 2 production.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Altered excitation energy transfer between phycobilisome and photosystems in the absence of ApcG, a small linker peptide, in Synechocystis sp. PCC 6803, a cyanobacterium

Phycobilisome (PBS) is a large pigment-protein complex in cyanobacteria and red algae responsible for capturing sunlight and transferring its energy to photosystems (PS). Spectroscopic and structural properties of various PBSs have been widely studied, however, the nature of so-called complex-complex interactions between PBS and PSs remains much less explored. In this work, we have investigated the function of a newly identified PBS linker protein, ApcG, some domain of which, together with a loop region (PB-loop in ApcE), is possibly located near the PBS-PS interface. Using Synechocystis sp. PCC 6803, we generated an ApcG deletion mutant and probed its deletion effect on the energetic coupling between PBS and photosystems. Steady-state and time-resolved spectroscopic characterization of the purified ΔApcG-PBS demonstrated that ApcG removal weakly affects the photophysical properties of PBS that the spectroscopic properties of terminal energy emitters are comparable to those of PBS from wild-type. However, analysis of fluorescence decay imaging datasets reveals that ApcG deletion induces disruptions within the allophycocyanin (APC) core, resulting in the emergence (splitting) of two spectrally diverse subgroups with some short-lived APC. Profound spectroscopic changes of the whole ΔApcG mutant cell, however, emerge during state transition, a dynamic process of light scheme adaptation. The mutant cells in State I show a substantial increase in PBS-related fluorescence. On the other hand, global analysis of time-resolved fluorescence demonstrates that in general ApcG deletion does not alter or inhibit state transitions if it is interpreted only in terms of the changes of the PSII and PSI fluorescence emission intensity. Furthermore, the results revealed yet–to–be discovered mechanism of ApcG-docking induced excitation energy transfer regulation within PBS or to Photosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Functional plasticity of HCO 3 – uptake and CO 2 fixation in Cupriavidus necator H16

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of β-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO 2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO 3 - uptake and CO 2 fixation in C. necator, providing new pathways for CO 2 -based biomanufacturing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Spatiotemporal 4D Whole-cell Modeling of a Minimal Autotroph Reveals Central Carbon Metabolism Regulated Locally by Protein Megacomplexes via Post-translational Modifications under Light Disturbance

Photosynthetic microorganisms rely on multiple pathways in central carbon metabolism to adapt to fluctuating light and energy availability across diel cycles. Mechanistic insight into the regulatory dynamics of this adaptation requires integrating processes spanning disparate timescales, from rapid redox-dependent post-translational modifications (PTMs) to slower changes in protein expression and metabolic pathway usage. To address this complexity beyond genome-based inference and traditional modeling, we develop a whole-cell four-dimensional (3D + time) model of the marine cyanobacterium Prochlorococcus marinus MED4 that explicitly represents the spatial organization of enzymatic and molecular processes in central carbon metabolism under light perturbation. We employ a perturbation-based research design to experimentally generate time-series, multi-omics measurements that provide molecular descriptors and cryo-ET derived 3D segmented volumes as constraints for this dynamic 4D framework. The integration of experiments and modeling across defined light regimes enables quantitative validation of system-level responses and forecasting under distinct light disturbances. We test the hypothesis that light-dependent redox PTMs regulating the structural assembly of a protein megacomplex, the “dark complex,” modulate metabolic flux at a conserved regulatory node of the Calvin–Benson cycle (CBC) in cyanobacteria. Our model shows that subcellular spatial organization buffers rapid light-induced changes in thylakoid reaction rates, which are followed by redox-PTM-mediated sequestration or release of CBC enzymes in the dark complex, ultimately impacting carbon fixation dynamics within carboxysomes. Comparison with an equivalently parameterized well-mixed stochastic model demonstrates that post-translational regulation not only buffers transcriptional noise and diffusion-driven fluctuations but also stabilizes phenotypic outcomes, underscoring the importance of spatial heterogeneity in phenotypic robustness. This ability to probe adaptive, spatiotemporally resolved mechanisms in photosynthetic machinery and central carbon metabolism addresses a critical gap in genotype-to-phenotype inference and expands modeling and design capabilities for understudied or genetically intractable autotrophs such as P. marinus MED4.

Johnson, Connah G.↗
DOE OSTI2022

Microcystin-LR aerosol induces inflammatory responses in healthy human primary airway epithelium

Harmful algal blooms plague bodies of freshwater globally. These blooms are often composed of outgrowths of cyanobacteria capable of producing the heptapeptide Microcystin-LR (MC-LR) which is a well-known hepatotoxin. Recently, MC-LR has been detected in aerosols generated from lake water. However, the risk for human health effects due to MC-LR inhalation exposure have not been extensively investigated. In this study, we exposed a fully differentiated 3D human airway epithelium derived from 14 healthy donors to MC-LR-containing aerosol for 3 minutes per day for 3 days. Concentrations of MC-LR ranged from 100 pM to 1 µM. Although there were little to no detrimental alterations in measures of the airway epithelial function (i.e. cell survival, tissue integrity, mucociliary clearance, or cilia beating frequency), a distinct shift in the transcriptional activity was found. Genes related to inflammation were found to be upregulated such as C-C motif chemokine 5 (CCL5; log2FC = 0.56 , p = 0.02) and C-C chemokine receptor type 7 (CCR7; log2FC = 0.83, p = 0.03). Functionally, conditioned media from MC-LR exposed airway epithelium was also found to have significant chemo-attractive properties for primary human neutrophils. Additionally, increases were found in the concentration of secreted chemokine proteins in the conditioned media such as CCL1 (log2FC = 5.07 , p = 0.0001) and CCL5 (log2FC = 1.02, p = 0.046). These results suggest that MC-LR exposure to the human airway epithelium is capable of inducing an inflammatory response that may potentiate acute or chronic disease.

3D human airway epithelium↗
DOE OSTI2020

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

1,2-propanediol utilization MCP↗
DOE OSTI2021

Photosynthetic adaptation to polar life: Energy balance, photoprotection and genetic redundancy

The persistent low temperature that characterize polar habitats combined with the requirement for light for all photoautotrophs creates a conundrum. The absorption of too much light at low temperature can cause an energy imbalance that decreases photosynthetic performance that has a negative impact on growth and can affect long-term survival. The goal of this review is to survey the mechanism(s) by which polar photoautotrophs maintain cellular energy balance, that is, photostasis to overcome the potential for cellular energy imbalance in their low temperature environments. Photopsychrophiles are photosynthetic organisms that are obligately adapted to low temperature (0⁰- 15 °C) but usually die at higher temperatures (≥20 °C). In contrast, photopsychrotolerant species can usually tolerate and survive a broad range of temperatures (5⁰- 40 °C). First, we summarize the basic concepts of excess excitation energy, energy balance, photoprotection and photostasis and their importance to survival in polar habitats. Second, we compare the photoprotective mechanisms that underlie photostasis and survival in aquatic cyanobacteria and green algae as well as terrestrial Antarctic and Arctic plants. We show that polar photopsychrophilic and photopsychrotolerant organisms attain energy balance at low temperature either through a regulated reduction in the efficiency of light absorption or through enhanced capacity to consume photosynthetic electrons by the induction of O 2 as an alternative electron acceptor. Finally, we compare the published genomes of three photopsychrophilic and one photopsychrotolerant alga with five mesophilic green algae including the model green alga, Chlamydomonas reinhardtii. We relate our genomic analyses to photoprotective mechanisms that contribute to the potential attainment of photostasis. Finally, we discuss how the observed genomic redundancy in photopsychrophilic genomes may confer energy balance, photoprotection and resilience to their harsh polar environment. Primary production in aquatic, Antarctic and Arctic environments is dependent on diverse algal and cyanobacterial communities. Although mosses and lichens dominate the Antarctic terrestrial landscape, only two extant angiosperms exist in the Antarctic. Furthermore, the identification of a single ‘molecular key’ to unravel adaptation of photopsychrophily and photopsychrotolerance remains elusive. Since these photoautotrophs represent excellent biomarkers to assess the impact of global warming on polar ecosystems, increased study of these polar photoautotrophs remains essential.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

How to correct relative voxel scale factors for calculations of vector-difference Fourier maps in cryo-EM

The atomic coordinates derived from cryo-electron microscopy (cryo-EM) maps can be inaccurate when the voxel scaling factors are not properly calibrated. Here, we describe a method for correcting relative voxel scaling factors between pairs of cryo-EM maps for the same or similar structures that are expanded or contracted relative to each other. We find that the correction of scaling factors reduces the amplitude differences of Fourier-inverted structure factors from voxel-rescaled maps by up to 20–30%, as shown by two cryo-EM maps of the SARS-CoV-2 spike protein measured at pH 4.0 and pH 8.0. This allows for the calculation of the difference map after properly scaling, revealing differences between the two structures for individual amino acid residues. Unexpectedly, the analysis uncovers two previously overlooked differences of amino acid residues in structures and their local structural changes. Furthermore, we demonstrate the method as applied to two cryo-EM maps of monomeric apo-photosystem II from the cyanobacteria Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus. The resulting difference maps reveal many changes in the peripheral transmembrane PsbX subunit between the two species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biological conversion of methane to methanol at high H 2 S concentrations with an H 2 S-tolerant methanotrophic consortium

To develop biological biogas to methanol conversion technology without costly hydrogen sulfide (H 2 S) removal, a high H 2 S tolerant methanotrophic consortium (HTMC), was enriched from an anaerobic digester effluent in presence of 5.64 g/m 3 H 2 S in the gas phase. The HTMC can grow stably and produce methanol under conditions with CH 4 /air mixtures containing 5.64 g/m 3 of H 2 S. There is no significant (p>0.05) difference in cell yield or CH 4 to methanol conversion efficiency between trials with different H 2 S concentrations from 0 g/m 3 to 5.64 g/m 3 . Further, under optimal conditions, a cell yield of 0.333 g cells/g CH 4 , a methanol concentration of 0.28 mg/mL, and a CH 4 to methanol conversion efficiency of 0.22 mol/mol were obtained, respectively. Besides methanotrophs (14.85%) and other bacteria, Cyanobacteria were also identified in the HTMC with a high abundancy (32.16%), which could broaden the application of HTMC for simultaneous utilization of CH 4 and CO 2 from raw biogas.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO 2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including E. coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate limiting steps in biological ethylene production. We employed a combination of genome scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to the EFE enzyme (WT vs mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Fluctuating pH for efficient photomixotrophic succinate production

Cyanobacteria are attracting increasing attention as a photosynthetic chassis organism for diverse biochemical production, however, photoautotrophic production remains inefficient. Photomixotrophy, a method where sugar is used to supplement baseline autotrophic metabolism in photosynthetic hosts, is becoming increasingly popular for enhancing sustainable bioproduction with multiple input energy streams. In this study, the commercially relevant diacid, succinate, was produced photomixotrophically. Succinate is an important industrial chemical that can be used for the production of a wide array of products, from pharmaceuticals to biopolymers. In this system, the substrate, glucose, is transported by a proton symporter and the product, succinate, is hypothesized to be transported by another proton symporter, but in the opposite direction. Thus, low pH is required for the import of glucose and high pH is required for the export of succinate. Succinate production was initiated in a pH 7 medium containing bicarbonate. Glucose was efficiently imported at around neutral pH. Utilization of bicarbonate by CO 2 fixation raised the pH of the medium. As succinate, a diacid, was produced, the pH of the medium dropped. By repeating this cycle with additional pH adjustment, those contradictory requirements for transport were overcome. pH affects a variety of biological factors and by cycling from high pH to neutral pH processes such as CO 2 fixation rates and CO 2 solubility can vary. In this study the engineered strains produced succinate during fluctuating pH conditions, achieving a titer of 5.0gL -1 after 10 days under shake flask conditions. Finally, these results demonstrate the potential for photomixotrophic production as a viable option for the large-scale production of succinate.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Identification of Serine-Containing Microcystins by UHPLC-MS/MS Using Thiol and Sulfoxide Derivatizations and Detection of Novel Neutral Losses

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria, and their structural diversity has led to the discovery of more than 300 congeners to date. However, with known amino acid combinations, many more MC congeners are theoretically possible, suggesting many remain unidentified. Herein, two novel serine (Ser)-containing MCs were putatively identified in a Lake Erie cyanobacterial harmful algal bloom (cyanoHAB), using high-resolution UHPLC-MS as well as thiol and sulfoxide derivatization procedures. These MCs contain an α,β-unsaturated carbonyl on methyl dehydroalanine (Mdha) residue that undergoes Michael addition to produce a thiol-derivatized MC. Derivatization reactions using various thiolation reagents were followed by MS/MS, and two Python codes were used for data analysis and structural elucidation of MCs. Two novel MCs containing Ser at position 1 (i.e., next to Mdha) were putatively identified as [Ser 1 ]MC-RR and [Ser 1 ]MC-YR. Using thiol- and sulfoxide-modified [Ser 1 ]MCs, identifications were confirmed by the observation of specific neutral losses of the oxidized thiols or sulfoxides in CID-MS/MS spectra in both positive and negative electrospray ionization (ESI) modes. These novel neutral losses are unique for MCs with Mdha and an adjacent Ser residue. In conclusion, data suggest that a gas-phase reaction occurs between oxygen from adjacent Ser residue and sulfur of the Mdha-bonded thiol or sulfoxide, which leads to the formation and detection of stable cyclic MC ions in MS/MS spectra at m/z values corresponding to the loss of oxidized thiols or oxidized sulfoxides from Ser 1 -containing MCs.

Premathilaka, Sanduni H.↗
DOE OSTI2023

Cyanobacterial Phycobilisome Allostery as Revealed by Quantitative Mass Spectrometry

Phycobilisomes (PBSs) are the major photosynthetic light-harvesting complexes in cyanobacteria and red algae. PBS, a multisubunit protein complex, has two major interfaces that comprise intrinsically disordered regions (IDRs): rod–core and core-membrane. IDRs do not form regular, three-dimensional structures on their own. Their presence in the photosynthetic pigment–protein complexes portends their structural and functional importance. A recent model suggests that PB-loop, an IDR located on the PBS subunit ApcE and C-terminal extension (CTE) of the PBS subunit ApcG, forms a structural protrusion on the PBS core-membrane side, facing the thylakoid membrane. Here, the structural synergy between the rod–core region and the core-membrane region was investigated using quantitative mass spectrometry (MS). The AlphaFold-predicted CpcG-CTE structure was first modeled onto the PBS rod–core region, guided and justified by the isotopically encoded structural MS data. Quantitative cross-linking MS analysis revealed that the structural proximity of the PB-loop in ApcE and ApcG-CTE is significantly disturbed in the absence of six PBS rods, which are attached to PBS via CpcG-CTE, indicative of drastic conformational changes and decreased structural integrity. Furthermore, these results suggest that CpcG-rod attachment on the PBS rod–core side is essentially required for the PBS core-membrane structural assembly. The hypothesized long-range synergy between the rod–core interface (where the orange carotenoid protein also functions) and the terminal energy emitter of PBS must have important regulatory roles in PBS core assembly, light-harvesting, and excitation energy transmission. These data also lend strategies that genetic truncation of the light-harvesting antennas aimed for improved photosynthetic productivity must rely on an in-depth understanding of their global structural integrity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Frequency-Domain Spectroscopic Study of the Photosystem I Supercomplexes, Isolated IsiA Monomers, and the Intact IsiA Ring

The PSI 3 -IsiA 18 supercomplex is one of the largest and most complicated assemblies in photosynthesis. The IsiA ring, composed of 18 IsiA monomers (IsiA 18 ) surrounding the PSI trimer (PSI 3 ), forms under iron-deficient condition in cyanobacteria and acts as a peripheral antenna. Based on the supercomplex structure recently determined via cryo-EM imaging, we model various optical spectra of the IsiA monomers and IsiA 18 ring. Comparison of the absorption and emission spectra of the isolated IsiA monomers and the full ring reveals that about 2.7 chlorophylls (Chls) are lost in the isolated IsiA monomers. The best fits for isolated monomers spectra are obtained assuming the absence of Chl 508, Chl 517 and 70% loss of Chl 511. The best model describing all three hexamers and the entire ring suggests that the lowest energy pigments are Chls 511, 514 and 517. Based on the modeling results presented in this work we conclude that there are most likely three entry points for EET from the IsiA6 hexamer to the PSI core monomer, with two of these entry points likely being located next to each other (i.e. nine entry points from IsiA 18 to the PSI 3 trimer). Lastly, we show that excitation energy transfer inside individual monomers is fast (< 2 ps at T = 5 K) and at least 20 times faster than inter-monomer energy transfer.

59 BASIC BIOLOGICAL SCIENCES↗