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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe 2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe 2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein–protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen–deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein–protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA–NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Structural and Biophysical Properties of a [4Fe-4S] Ferredoxin-Like Protein from Synechocystis sp. PCC 6803 with a Unique Two Domain Structure

Electron carrier proteins (ECPs), binding iron-sulfur clusters, are vital components within the intricate network of metabolic and photosynthetic reactions. They play a crucial role in the distribution of reducing equivalents. In Synechocystis sp. PCC 6803, the ECP network includes at least nine ferredoxins. Previous research, including global expression analyses and protein binding studies, has offered initial insights into the functional roles of individual ferredoxins within this network. This study primarily focuses on Ferredoxin 9 (slr2059). Through sequence analysis and computational modeling, Ferredoxin 9 emerges as a unique ECP with a distinctive two-domain architecture. It consists of a C-terminal iron-sulfur binding domain and an N-terminal domain with homology to Nil-domain proteins, connected by a structurally rigid 4-amino acid linker. Notably, in contrast to canonical [2Fe-2S] ferredoxins exemplified by PetF (ssl0020), which feature highly acidic surfaces facilitating electron transfer with photosystem I reaction centers, models of Ferredoxin 9 reveal a more neutral to basic protein surface. Using a combination of electron paramagnetic resonance spectroscopy and square-wave voltammetry on heterologously produced Ferredoxin 9, this study demonstrates that the protein coordinates 2x[4Fe-4S]2+/1+ redox-active and magnetically interacting clusters, with measured redox potentials of -420 +/- 9 mV and -516 +/- 10 mV vs SHE. A more in-depth analysis of Fdx9's unique structure and protein sequence suggests that this type of Nil-2[4Fe-4S] multi-domain ferredoxin is well conserved in cyanobacteria, bearing structural similarities to proteins involved in homocysteine synthesis in methanogens.

cyanobacteria↗
DOE OSTI2021

Quality control of mislocalized and orphan proteins

A healthy and functional proteome is essential to cell physiology. However, this is constantly being challenged as most steps of protein metabolism are error-prone and changes in the physico-chemical environment can affect protein structure and function, thereby disrupting proteome homeostasis. Among a variety of potential mistakes, proteins can be targeted to incorrect compartments or subunits of protein complexes may fail to assemble properly with their partners, resulting in the formation of mislocalized and orphan proteins, respectively. Quality control systems are in place to handle these aberrant proteins, and to minimize their detrimental impact on cellular functions. Here, we discuss recent findings on quality control mechanisms handling mislocalized and orphan proteins. We highlight common principles involved in their recognition and summarize how accumulation of these aberrant molecules is associated with aging and disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria

Synechococcus elongatus PCC 7942 is a model cyanobacterium for study of the circadian clock, photosynthesis, and bioproduction of chemicals, yet nearly 40% of its gene identities and functions remain unknown, in part due to limitations of the existing genetic toolkit. While classical techniques for the study of genes (e.g., deletion or mutagenesis) can yield valuable information about the absence of a gene and its associated protein, there are limits to these approaches, particularly in the study of essential genes. In this research, we developed a tool for inducible degradation of target proteins in S. elongatus by adapting a method using degron tags from the Mesoplasma florum transfer-messenger RNA (tmRNA) system. We observed that M. florum lon protease can rapidly degrade exogenous and native proteins tagged with the cognate sequence within hours of induction. We used this system to inducibly degrade the essential cell division factor, FtsZ, as well as shell protein components of the carboxysome. Our results have implications for carboxysome biogenesis and the rate of carboxysome turnover during cell growth. Lon protease control of proteins offers an alternative approach for the study of essential proteins and protein dynamics in cyanobacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Incorporation of sensing modalities into de novo designed fluorescence-activating proteins

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis -planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca 2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein–protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

De novo design of modular protein hydrogels with programmable intra- and extracellular viscoelasticity

Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Engineering the green algae Chlamydomonas incerta for recombinant protein production

Chlamydomonas incerta , a genetically close relative of the model green alga Chlamydomonas reinhardtii , shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii , this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta ’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii , C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii . For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii , as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta ’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

cell membranes↗
NASA NTRS2002

Expression and affinity purification of recombinant proteins from plants

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

Non-NASA Center↗
DOE OSTI2023

A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Scalable, two–stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed–batch fermentations enables cell densities of ~30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (~270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μl (384–well plates), 100 μl (96–well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ~5–7 gCDW/L, offering protein titers above 2 g/L. Here, the methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed–batch fermentation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Examining polymer‐protein biophysical interactions with small‐angle x‐ray scattering and quartz crystal microbalance with dissipation

Abstract Polymer‐protein hybrids can be deployed to improve protein solubility and stability in denaturing environments. While previous work used robotics and active machine learning to inform new designs, further biophysical information is required to ascertain structure–function behavior. Here, we show the value of tandem small‐angle x‐ray scattering (SAXS) and quartz crystal microbalance with dissipation (QCMD) experiments to reveal detailed polymer‐protein interactions with horseradish peroxidase (HRP) as a test case. Of particular interest was the process of polymer‐protein complex formation under thermal stress whereby SAXS monitors formation in solution while QCMD follows these dynamics at an interface. The radius of gyration ( R g ) of the protein as measured by SAXS does not change significantly in the presence of polymer under denaturing conditions, but thickness and dissipation changes were observed in QCMD data. SAXS data with and without thermal stress were utilized to create bead models of the potential complexes and denatured enzyme, and each model fit provided insight into the degree of interactions. Additionally, QCMD data demonstrated that HRP deforms by spreading upon surface adsorption at low concentration as shown by longer adsorption times and smaller frequency shifts. In contrast, thermally stressed and highly inactive HRP had faster adsorption kinetics. The combination of SAXS and QCMD serves as a framework for biophysical characterization of interactions between proteins and polymers which could be useful in designing polymer‐protein hybrids.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics

Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and applied it to screen 61eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functional competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatrices

Sensitive, multiplexed protein quantification remains challenging despite recent advancements in LC-MS assays for targeted protein biomarker quantification. High-sensitivity protein biomarker measurements usually require immuno-affinity enrichment of target protein; a process which is highly dependent on capture reagent and limited in capability to measure multiple analytes. Herein, we report a novel antibody-free platform, which measures multiple biomarkers from complex matrices employing a strategically optimized solid-phase extraction cleanup and orthogonal multidimensional LC-MS. Eight human protein biomarkers with different specifications were spiked into canine plasma as a model investigation system. The developed strategy achieved the desired sensitivity, robustness, and throughput via the following steps: (1) post digestion mixed-mode cation exchange-reverse phase SPE enrichment cleaned up the sample initially; (2) rapid, high-pH peptide fractionation further eliminated background components efficiently while selectively enriched signature peptides (SP) to provide sufficient sensitivity for multiple targets; and (3) trapping-micro-LC-MS analysis delivered high sensitivity comparable to a nano-LC-MS method but with much better robustness and throughput for the final analysis. Compared with a conventional LC-MS assay with direct protein digestion and limited clean-up, analysis with this antibody-free platform improved the LLOQ by 1–2 orders of magnitude for the eight protein biomarkers, reaching as low as 5 ng/mL in plasma, with feasible robustness and throughput. In conclusion, this platform was applied for the quantification of biomarkers of respiratory conditions in patients with various lung diseases, demonstrating real-world applicability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.

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

Cross-reactive immunogenicity of group A streptococcal vaccines designed using a recurrent neural network to identify conserved M protein linear epitopes

The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal sequence of the protein defines the more than 200 M types of Strep A and also contains epitopes that elicit opsonic antibodies, some of which cross-react with heterologous M types. Current efforts to develop broadly protective M protein-based vaccines are directed at identifying potential cross-protective epitopes located in the N-terminal regions of cluster-related M proteins for use as vaccine antigens. In this study, we have used a comprehensive approach using the recurrent neural network ABCpred and IEDB epitope conservancy analysis tools to predict 16 residue linear B-cell epitopes from 117 clinically relevant M types of Strep A (~88% of global Strep A infections). Furthermore, to examine the immunogenicity of these epitope-based vaccines, nine peptides that together shared ≥60% sequence identity with 37 heterologous M proteins were incorporated into two recombinant hybrid protein vaccines, in which the epitopes were repeated 2 or 3 times, respectively. The combined immune responses of immunized rabbits showed that the vaccines elicited significant levels of antibodies against all nine vaccine epitopes present in homologous N-terminal 1–50 amino acid synthetic M peptides, as well as cross-reactive antibodies against 16 of 37 heterologous M peptides predicted to contain similar epitopes. The epitope-specificity of the cross-reactive antibodies was confirmed by ELISA inhibition assays and functional opsonic activity was assayed in HL-60-based bactericidal assays. The results provide important information for the future design of broadly protective M protein-based Strep A vaccines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Protein Coatings Dictate the Dispersibility and Stability of Hydrophobic Zeolitic-Imidazolate Frameworks in Water

Metal–organic frameworks are promising materials for many biomedical technologies due to their ability to store and release large quantities of guest molecules in a predictable and tunable fashion. In biological fluids, proteins readily adsorb to the external surfaces of metal–organic framework particles through a combination of hydrophobic and electrostatic interactions. However, much remains to be understood about the nature of these protein coatings and how they influence the bulk properties of aqueous dispersions of metal–organic frameworks. Here, in this work, we show that a variety of proteins can be used to manipulate the properties of aqueous dispersions of zeolitic-imidazolate framework (ZIF) particles. Specifically, noncovalently associated protein coatings promote the formation of dispersions of hydrophobic ZIFs in water with high colloidal and hydrolytic stability, as long as the density of adsorbed proteins exceeds a critical, protein-dependent threshold. Further, these dispersions feature low viscosity and complete retention of gas carrying capacity. The wide range of properties accessible with protein coatings provides a highly modular approach to design hydrophobic metal–organic frameworks with properties tailored for specific biological applications.

adsorption↗
DOE OSTI2022

De novo protein design of photochemical reaction centers

Abstract Natural photosynthetic protein complexes capture sunlight to power the energetic catalysis that supports life on Earth. Yet these natural protein structures carry an evolutionary legacy of complexity and fragility that encumbers protein reengineering efforts and obfuscates the underlying design rules for light-driven charge separation. De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build new enzymes for efficient solar-to-fuel energy conversion. Here, we report the rational design, X-ray crystal structure, and electron transfer activity of a multi-cofactor protein that incorporates essential elements of photosynthetic reaction centers. This highly stable, modular artificial protein framework can be reconstituted in vitro with interchangeable redox centers for nanometer-scale photochemical charge separation. Transient absorption spectroscopy demonstrates Photosystem II-like tyrosine and metal cluster oxidation, and we measure charge separation lifetimes exceeding 100 ms, ideal for light-activated catalysis. This de novo-designed reaction center builds upon engineering guidelines established for charge separation in earlier synthetic photochemical triads and modified natural proteins, and it shows how synthetic biology may lead to a new generation of genetically encoded, light-powered catalysts for solar fuel production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Directing polymorph specific calcium carbonate formation with de novo protein templates

Biomolecules modulate inorganic crystallization to generate hierarchically structured biominerals, but the atomic structure of the organic-inorganic interfaces that regulate mineralization remain largely unknown. We hypothesized that heterogeneous nucleation of calcium carbonate could be achieved by a structured flat molecular template that pre-organizes calcium ions on its surface. To test this hypothesis, we design helical repeat proteins (DHRs) displaying regularly spaced carboxylate arrays on their surfaces and find that both protein monomers and protein-C a2+ supramolecular assemblies directly nucleate nano-calcite with non-natural {110} or {202} faces while vaterite, which forms first in the absence of the proteins, is bypassed. These protein-stabilized nanocrystals then assemble by oriented attachment into calcite mesocrystals. We find further that nanocrystal size and polymorph can be tuned by varying the length and surface chemistry of the designed protein templates. Thus, bio-mineralization can be programmed using de novo protein design, providing a route to next-generation hybrid materials.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗