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64 records · Page 4

All sources NASA NTRS DOE OSTI

DOE OSTI2020

Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO 2 -concentrating mechanism by facilitating high CO 2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein–protein interactions that drive the assembly process. In this work, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Enhanced reactivity at the oil–water interface accelerates the synthesis of zymonic acid in microemulsions

Chemical reactions in microscale compartments, such as aerosols and emulsions, can exhibit significantly faster reaction rates relative to macroscale containers. This enhancement in chemistry is often due to the elevated importance of surfaces as reaction vessels are reduced to picoliter volumes. While most studies have focused on the air-water interface of droplets, there are comparably fewer studies of reactions in micron-scale aqueous solutions encapsulated by oil. Here we investigate the condensation reaction of pyruvic acid (PA) to form zymonic acid (ZA) and water. Using microfluidics and optical trapping, chemical kinetics are measured in monodisperse micron-sized emulsion droplets in situ via Raman spectroscopy. Relative to a macroscopic bulk solution, which exhibits little to no reaction over many days, we find efficient production of ZA over the same time period. A kinetic model is developed to elucidate the role of the interface in accelerating the microdroplet reaction kinetics. After quantifying the surface partitioning of PA from interfacial tension measurements, the rate coefficient for the condensation reaction at the oil-water interface is determined to be 1.8 × 10-2 M-1 s-1. This rate coefficient is estimated to be 105 larger than the reaction rate in bulk aqueous solutions. Compared to previous studies of accelerated ZA formation at the air-water interface on nanodroplets, we find that the reaction at the oil-water interface is 20 times more efficient. Despite this difference, the overall ZA formation rate in emulsions is significantly slower than in the same-sized aerosols, which arises from the weaker partitioning of PA to the oil-aqueous relative to air-water interface. These results highlight the interplay between interfacial partitioning and reactivity in accelerating chemistry in microcompartments and provides new insights into how interfacial composition influences condensation reactions.

Wallace, Brandon J↗

DOE OSTI2025

Quantum sensing of paramagnetic analytes by nanodiamonds in levitated microdroplets and aqueous solutions

Nanodiamonds (ND) hosting negatively charged nitrogen-vacancy (NV-) color centers have received attention for applications in magnetic field, electric field, chemical, and bio-sensing. The versatility of these probes is their excellent room-temperature optical and spin properties, along with their small size, functionalized surfaces and resistance to bleaching, making them ideal as nanoscopic sensors in picoliter volumes (e.g. single cells, but also microcompartments and aerosols). For quantitative ND-NV- sensing of paramagnetic analytes in such contexts, however, there remains an incomplete understanding of how factors related to the aqueous phase environment control detection efficiency. To address this, optically detected magnetic resonance (ODMR) is measured in bulk macroscale solutions and single levitated microdroplets as a function of Gd+3 concentration (340 nM to 1.5 mM), nanodiamond size, pH, competitor ions, and ligands. The ODMR response to [Gd+3] is found to be nonlinear, and pH, ND and sample volume dependent; indicating the detection of Gd+3 requires efficient adsorption of the analyte to the diamond surface. Langmuir adsorption isotherms embedded in a quantitative photophysical model links the ODMR response to adsorption thermodynamics of Gd+3. The equilibrium constant for Gd+3 adsorption to a carboxylated ND surface is determined to be (1 ± 0.5) x 105 M-1 corresponding to a free energy of adsorption of (-28 ± 1) kJ mol-1. These results provide general insight into how complex aqueous and microscale environments impact nanodiamond based quantum sensing modalities, and portend their application as quantitative chemical sensors in microenvironments.

Brown, Emily K↗

DOE OSTI2022

Role of carboxysomes in cyanobacterial CO 2 assimilation: CO 2 concentrating mechanisms and metabolon implications

Many carbon-fixing organisms have evolved CO 2 concentrating mechanisms (CCMs) to enhance the delivery of CO 2 to RuBisCO, while minimizing reactions with the competitive inhibitor, molecular O 2 . These distinct types of CCMs have been extensively studied using genetics, biochemistry, cell imaging, mass spectrometry, and metabolic flux analysis. Highlighted in this paper, the cyanobacterial CCM features a bacterial microcompartment (BMC) called ‘carboxysome’ in which RuBisCO is co-encapsulated with the enzyme carbonic anhydrase (CA) within a semi-permeable protein shell. Further, the cyanobacterial CCM is capable of increasing CO 2 around RuBisCO, leading to one of the most efficient processes known for fixing ambient CO 2 . The carboxysome life cycle is dynamic and creates a unique subcellular environment that promotes activity of the Calvin–Benson (CB) cycle. The carboxysome may function within a larger cellular metabolon, physical association of functionally coupled proteins, to enhance metabolite channelling and carbon flux. In light of CCMs, synthetic biology approaches have been used to improve enzyme complex for CO 2 fixations. Research on CCM-associated metabolons has also inspired biologists to engineer multi-step pathways by providing anchoring points for enzyme cascades to channel intermediate metabolites towards valuable products.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Plastoquinone redox status influences carboxysome integrity via a RpaA ‐ and reactive oxygen species‐dependent regulatory network

SUMMARY Carboxysomes are bacterial microcompartments that encapsulate Rubisco and are a core component of the cyanobacterial carbon concentration mechanism (CCM). While carboxysome number, size, and spatial organization vary in different environmental conditions (CO 2 , light availability, redox state, temperature, and light quality), the molecular mechanisms underlying this potentially adaptive process remain elusive. Herein, we observe that mutants of the circadian rhythm/metabolism factor, Regulator of Phycobilisome Association A (RpaA), exhibit a striking breakdown of carboxysomes under certain environmental conditions. We find that conditions leading to overreduction of the plastoquinone (PQ) pool (mixotrophic growth, high irradiance, or chemical inhibition of electron transfer from PQ to the cytochromeb 6 fcomplex) are accompanied by an elevated generation of reactive oxygen species (ROS) and correlate with the loss of carboxysome integrity. Carboxysome breakdown is reversed by environmental conditions or chemical inhibitors that prevent PQ overreduction and accompanying ROS generation. Taken together, our data support a novel link between the redox status of the PQ pool and carboxysome integrity. Our results have implications for the fundamental understanding of cyanobacterial energy‐balancing pathways and may indicate new research directions for understanding how the carboxysome is remodeled in response to changing environments.

Plant Sciences↗

DOE OSTI2020

Life cycle of a cyanobacterial carboxysome

Carboxysomes, prototypical bacterial microcompartments (BMCs) found in cyanobacteria, are large (~1 GDa) and essential protein complexes that enhance CO 2 fixation. While carboxysome biogenesis has been elucidated, the activity dynamics, lifetime, and degradation of these structures have not been investigated, owing to the inability of tracking individual BMCs over time in vivo. In this work, we have developed a fluorescence-imaging platform to simultaneously measure carboxysome number, position, and activity over time in a growing cyanobacterial population, allowing individual carboxysomes to be clustered on the basis of activity and spatial dynamics. We have demonstrated both BMC degradation, characterized by abrupt activity loss followed by polar recruitment of the deactivated complex, and a subclass of ultraproductive carboxysomes. Together, our results reveal the BMC life cycle after biogenesis and describe the first method for measuring activity of single BMCs in vivo.

09 BIOMASS FUELS↗

DOE OSTI2021

Carbon isotope evidence for the global physiology of Proterozoic cyanobacteria

Ancestral cyanobacteria are assumed to be prominent primary producers after the Great Oxidation Event [≈2.4 to 2.0 billion years (Ga) ago], but carbon isotope fractionation by extant marine cyanobacteria (α-cyanobacteria) is inconsistent with isotopic records of carbon fixation by primary producers in the mid-Proterozoic eon (1.8 to 1.0 Ga ago). To resolve this disagreement, we quantified carbon isotope fractionation by a wild-type planktic β-cyanobacterium (Synechococcus sp. PCC 7002), an engineered Proterozoic analog lacking a CO 2 -concentrating mechanism, and cyanobacterial mats. At mid-Proterozoic pH and pCO 2 values, carbon isotope fractionation by the wild-type β-cyanobacterium is fully consistent with the Proterozoic carbon isotope record, suggesting that cyanobacteria with CO 2 -concentrating mechanisms were apparently the major primary producers in the pelagic Proterozoic ocean, despite atmospheric CO 2 levels up to 100 times modern. The selectively permeable microcompartments central to cyanobacterial CO 2 -concentrating mechanisms (“carboxysomes”) likely emerged to shield rubisco from O 2 during the Great Oxidation Event.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Inferring assembly-curving trends of bacterial micro-compartment shell hexamers from crystal structure arrangements

Bacterial microcompartments (BMC) are complex macromolecular assemblies that participate in varied chemical processes in about one fourth of bacterial species. BMC-encapsulated enzymatic activities are segregated from other cell contents by means of semipermeable shells, justifying why BMC are viewed as prototype nano-reactors for biotechnological applications. Herein, we undertook a comparative study of bending propensities of BMC hexamers (BMC-H), the most abundant shell constituents. Published data show that some BMC-H, like β -carboxysomal CcmK, tend to assemble flat whereas other BMC-H often build curved objects. Inspection of available crystal structures presenting BMC-H in tiled arrangements permitted us to identify two major assembly modes with a striking connection with experimental trends. All-atom molecular dynamics (MD) supported that BMC-H bending is triggered robustly only from the arrangement adopted in crystals by BMC-H that experimentally form curved objects, leading to very similar arrangements to those found in structures of recomposed BMC shells. Simulations on triplets of planar-behaving hexamers, which were previously reconfigured to comply with such organization, confirmed that bending propensity is mostly defined by the precise lateral positioning of hexamers, rather than by BMC-H identity. Finally, an interfacial lysine was pinpointed as the most decisive residue in controlling PduA spontaneous curvature. Globally, results presented herein should contribute to improve our understanding of the variable mechanisms of biogenesis characterized for BMC, and of possible strategies to regulate BMC size and shape.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1998

Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

Non-NASA Center↗

DOE OSTI2025

Robust Synthetic Biology Toolkit to Advance Carboxysome Study and Redesign

Carboxysomes are polyhedral protein organelles that microorganisms use to facilitate carbon dioxide assimilation. They are composed of a modular protein shell that envelops an enzymatic core mainly composed of physically coupled Rubisco and carbonic anhydrase. While the modular construction principles of carboxysomes make them attractive targets as customizable metabolic platforms, their size and complexity can be a hindrance. In this work, we design and validate a plasmid set, the pXpressome toolkit, in which α-carboxysomes are robustly expressed and remain intact and functional after purification. We tested this toolkit by introducing mutations that influence carboxysome structure and performance. We find that deletion of vertex-capping genes results in formation of larger carboxysomes, while deletion of facet forming genes produces smaller particles, suggesting that adjusting the ratio of these proteins can rationally affect morphology. Through a series of fluorescently labeled constructs, we observe that this toolkit leads to more uniform expression and better cell health than previously published carboxysome expression systems. Overall, the pXpressome toolkit facilitates the study and redesign of carboxysomes with robust performance and improved phenotype uniformity. The pXpressome toolkit will support efforts to remodel carboxysomes for enhanced carbon fixation or serve as a platform for other nanoencapsulation goals.

59 BASIC BIOLOGICAL SCIENCES↗