Results for “protein delivery” — Page 7

DOE OSTI2025

Nanolipoprotein particle (NLP) vaccine confers protection against Yersinia pestis aerosol challenge in a BALB/c mouse model

Introduction: Yersinia pestis is the etiological agent of plague, a disease that remains a concern as demonstrated by recent outbreaks in Madagascar. Infection with Y. pestis results in a rapidly progressing illness that can only be successfully treated with antibiotics given shortly after symptom onset. Live attenuated or whole cell inactivated vaccines confer protection against bubonic plague, but pneumonic plague has been more difficult to prevent. Novel effective subunit vaccine formulations may circumvent some of these shortfalls. Here, we compare the immunogenicity generated by an advanced subunit vaccine (F1V fusion protein) and a nanolipoprotein particle (NLP)-based vaccine. Methods: The NLP, a high-density lipoprotein mimetic, provides a nanoscale delivery platform for recombinant Y. pestis antigens LcrV (V) and F1. BALB/c mice were immunized via subcutaneous injection twice, three or four weeks apart. Four weeks later, splenocytes and sera were collected for immune profiling, and mice were challenged with aerosolized Y. pestis CO92. Results: Both formulations induced a strong IgG response against the F1 and V proteins, along with a robust memory B cell response and a balanced cell-mediated immune response as evidenced by both Th1- and Th2-related cytokines. The NLP-based vaccine induced a stronger cytokine response against F1, V, and F1V proteins relative to the F1V vaccine. As with F1V, the inclusion of Alhydrogel (Alu) in NLP vaccine formulations was critical for enhanced immunogenicity and protective efficacy. Mice that received two doses of F1:V:NLP + Alu and CpG were completely protected from a challenge with approximately eight median lethal doses of aerosolized Y. pestis CO92 and this protection confirmed the well-documented synergy between the F1 and V antigens in context of pneumonic plague. The NLPs have defined regions of polarity that facilitates the incorporation of a wide range of adjuvants and antigens with distinct physicochemical properties and are an excellent candidate platform for the development of multi-antigen vaccines.

F1↗

DOE OSTI2021

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Protein 4.1R affects photodynamic therapy for B16 melanoma by regulating the transport of 5-aminolevulinic acid

Highlights: • Protein 4.1R knockout promoted the proliferation, migration, and invasion of B16 cells. • Protein 4.1R knockout induced a decrease expression of 5-ALA transporters GAT-1 and GAT-2. • Protein 4.1R knockout reduced the effect of PDT and downregulated the anti-tumor immune response induced by PDT. Melanoma is the most aggressive malignant tumor of skin cancer as it can grow rapidly and metastasize. Photodynamic therapy (PDT) is a promising cancer ablation method for skin tumors, although it lacks efficiency owing to factors such as tumor characteristics, delivery of photosensitizers, immune response in vivo etc. Extensive investigation of molecules that can potentially modulate treatment efficacy is required. Protein 4.1R is a cytoskeletal protein molecule. Previous studies have shown that protein 4.1R knockdown reduces PDT sensitivity in mouse embryonic fibroblast cells. However, the functional role of protein 4.1R in melanoma is unclear. In this study, we aimed to elucidate the effect of protein 4.1R on PDT for melanoma in mice and the mechanism of anti-tumor immunity. Our results indicated that CRISPR/Cas9-mediated protein 4.1R knockout promotes the proliferation, migration, and invasion of B16 cells. We further investigated the potential mechanism of protein 4.1R on tumor cell PDT sensitivity. Our results showed that protein 4.1R knockout reduced the expression of membrane transporters γ-aminobutyric acid transporter (GAT)-1 and (GAT)-2 in B16 cells, which affected 5-ALA transmembrane transport and reduced the efficiency of PDT on B16 cells. Protein 4.1R knockout downregulated the anti-tumor immune response triggered by PDT in vivo. In conclusion, our data suggest that protein 4.1R is an important regulator in PDT for tumors and may promote the progress and efficacy of melanoma treatment.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site

Background: Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. Results: Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H 2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. Conclusion: The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O 2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs

μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the μNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of μNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448–605 of μNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-μNS (448–605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-μNS (448–605) baculovirus formed rod-shaped microcrystals (5–15 µm in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Å resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of μNS (448–605) , the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the μNS protein.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

The Detection of Aromatic Amino Acids in CR Chondrites Suggests They are Prebiotically Plausible

Amino acids (AAs) are the building blocks of proteins and are essential for life on Earth. Sources of AAs on early Earth range from exogenous sources such as delivery from extraterrestrial materials(i.e., meteorites, interplanetary dust particles, comets, etc.) to endogenous sources including chemical reactions in hydrothermal vents or atmospheric mixtures. Analyses of AAs of extraterrestrial origin from meteorites and laboratory studies of simulated prebiotic environments revealed that a diverse set of abiotically produced AAs may have been present on early Earth. However only a fraction of these AAs is found within the proteins of living organisms. Of the22 genetically encoded (proteinogenic) AAs, 20 occur in the standard genetic code (SGC) and are used by nearly all living organisms to build proteins. The universality of this AA alphabet suggests the presence and incorporation of these specific AAs occurred early in the evolution of life.

A. S. Burton↗

DOE OSTI2021

Serial femtosecond and serial synchrotron crystallography can yield data of equivalent quality: A systematic comparison

For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution.

59 BASIC BIOLOGICAL SCIENCES↗

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 OSTI2022

Alphavirus Particles Can Assemble with an Alternate Triangulation Number

Alphaviruses are spherical, enveloped RNA viruses primarily transmitted by mosquitoes, and cause significant arthritogenic and neurotropic disease in humans and livestock. Previous reports have shown that—in contrast to prototypical icosahedral viruses—alphaviruses incorporate frequent defects, and these may serve important functions in the viral life cycle. We confirm the genus-wide pleomorphism in live viral particles and extend our understanding of alphavirus assembly through the discovery of an alternate architecture of Eastern equine encephalitis virus (EEEV) particles. The alternate T = 3 icosahedral architecture differs in triangulation number from the classic T = 4 icosahedral organization that typifies alphaviruses, but the alternate architecture maintains the quasi-equivalence relationship of asymmetric units. The fusion spike glycoproteins are more loosely apposed in the T = 3 form with corresponding changes in the underlying capsid protein lattice. This alternate architecture could potentially be exploited in engineering alphavirus-based particles for delivery of alphaviral or other RNA.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Impact of Surface Chemistry of Ultrasmall Superparamagnetic Iron Oxide Nanoparticles on Protein Corona Formation and Endothelial Cell Uptake, Toxicity, and Barrier Function

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) have been investigated for biomedical applications, including novel contrast agents, magnetic tracers for tumor imaging, targeted drug delivery vehicles, and magneto-mechanical actuators for hyperthermia and thrombolysis. Despite significant progress, recent clinical reports have raised concerns regarding USPION safety related to endothelial cell dysfunction; however, there is limited information on factors contributing to these clinical responses. The influence of USPION surface chemistry on nanoparticle interactions with proteins may impact endothelial cell function leading to adverse responses. Therefore, the goal of this study was to assess the effects of carboxyl-functionalized USPION (CU) or amine-functionalized USPION (AU) (approximately 30 nm diameter) on biological responses in human coronary artery endothelial cells. Increased protein adsorption was observed for AU compared with CU after exposure to serum proteins. Exposure to CU, but not AU, resulted in a concentration-dependent decrease in cell viability and perinuclear accumulation inside cytoplasmic vesicles. Internalization of CU was correlated with endothelial cell functional changes under non-cytotoxic conditions, as evidenced by a marked decreased expression of endothelial-specific adhesion proteins (eg, vascular endothelial-cadherin and platelet endothelial cell adhesion molecule-1) and increased endothelial permeability. Evaluation of downstream signaling indicated endothelial permeability is associated with actin cytoskeleton remodeling, possibly elicited by intracellular events involving reactive oxygen species, calcium ions, and the nanoparticle cellular uptake pathway. This study demonstrated that USPION surface chemistry significantly impacts protein adsorption and endothelial cell uptake, viability, and barrier function. This information will advance the current toxicological profile of USPION and improve development, safety assessment, and clinical outcomes of USPION-enabled medical products.

Toxicology↗

DOE OSTI2020

Tubulin Double Helix: Lateral and Longitudinal Curvature Changes of Tubulin Protofilament

By virtue of their native structures, tubulin dimers are protein building blocks that are naturally pre-programmed to assemble into cytoskeletal polymers known as microtubules (MTs). Here we demonstrate polycation-directed (i.e. electrostatically tunable) assembly of tubulins through tubulin protofilament conformational changes in the longitudinal and lateral directions, creating novel tubulin double helices and various tubular architectures. Synchrotron small angle X-ray scattering and transmission electron microscopy reveal a remarkable range of nanoscale assembly structures: single- and double-layered double-helix tubulin tubules. The phase transitions from MTs into the new assemblies are dependent on the size and concentration of polycations. Two characteristic scales that determine the number of observed phases are the size of polycation compared to the size of tubulin (≈4 nm) and MT diameter (≈25 nm). This work suggests the feasibility of “programmable breakdown” of protein nanotubes, tearing MTs into double-stranded tubulins and building up previously undiscovered nanostructures, by using polycations with scissor- and glue-like properties. Importantly, we define a new role of tubulins as two-dimensionally shape-controllable building blocks for novel supramolecular architectures. Furthermore, these findings provide insight into the design of protein-based functional materials, for example, as metallization templates for nanoscale electronic devices, kinesin motor-driving molecular screws, and anticancer drug delivery vehicles.

2D shape‐control↗

NASA NTRS2015

Biomaterials Out of Thin Air: in Situ, On-Demand Printing of Advanced Biocomposites

Upmass is the single most significant limitation of our current space mission capability. Although biomaterials and biocomposites have mass, strength, flexibility, and self-healing properties that could significantly reduce upmass, their use is limited by the following drawbacks: Expensive, specific production. Many biomaterials can only be produced as part of significant support ecosystem; Inaccessible functional customization. The grain of wood, the porosity of bone, and so on are an integral part of the materials' desired mechanical properties, but are not deterministic when the material is naturally grown; Limited compositions. Most biomaterials (unlike metal, plastic, etc.) cannot be easily combined or modified to produce new materials. This project builds on recent advances in: Synthetic biology. Libraries of standardized genetic parts which can be used for controlled cellular material production, delivery, and binding; 3D printing. Commercial off-the-shelf components which can be used to make of a pico- to nanoliter cell deposition system; Tissue engineering. Proven cell-compatible support hydrogels and scaffolds can be modified to bind the deposited biomaterials of interest. Objectives: Feasibility and benefit analysis. Two mission contexts span the concept's scope (see below); Proof-of-concept demonstration. A simple grid of two proteins, fluorescent for easy detection, to validate the core technology concept; Proposed implementations for follow-on work. Avenues for future work on each core component (host cell, production control, material delivery, material binding, etc.); Complementary studies exploration. A survey of other emerging areas (in situ resource utilization, protein engineering, etc.) with the potential to multiply our technology's impact. Potential Impacts: This application could dramatically expand manufacturing capabilities on Earth and in space: In situ resource utilization. A far greater range of materials and products will be available from the limited palette offered by in situ resource extraction techniques; Reduced equipment and material upmass for off-Earth habitats. Ready- to-use highly specialized construction materials (radiation hardened, compressive/tensile, light or dense) from an extremely low starting mass; Structured biomaterial production. New ready-to-use macro, micro, and molecular manufacturing techniques for traditional biomaterials such as wood, bone and shell; New and novel biocomposite creation. The ability to create completely novel material composites from any base material that cells can be engineered to produce. Suggested Mission Contexts: ISS part manufacturing. A 'minimal working example' making a finished biomaterial part aboard the International Space Station; A long-term Mars habitat. 'Cradle-to-grave' use at a hypothetical Mars habitat, covering everything from tools to construction materials. Alternate Abstract: Imagine being able to print anything from tools and composite building materials to food and human tissues. Imagine being on Mars with the ability to replace any broken part, whether it's a part of your spacesuit, your habitat, or your own body. We propose a technique that would allow just that. By printing 3D arrays of cells engineered to secrete the necessary materials, the abundant in situ resources of atmosphere and regolith become organic, inorganic, or organic-inorganic composite materials. Such materials include novel, biologically derived materials not previously possible to fabricate.

habitats↗

NASA NTRS2015

Biomaterials Out of Thin Air: in Situ, On-demand Printing of Advanced Biocomposites

Upmass is the single most significant limitation of our current space mission capability. Although biomaterials and biocomposites have mass, strength, flexibility, and self-healing properties that could significantly reduce upmass, their use is limited by the following drawbacks: Expensive, specific production. Many biomaterials can only be produced as part of significant support ecosystem; Inaccessible functional customization. The grain of wood, the porosity of bone, and so on are an integral part of the materials' desired mechanical properties, but are not deterministic when the material is naturally grown; Limited compositions. Most biomaterials (unlike metal, plastic, etc.) cannot be easily combined or modified to produce new materials. This project builds on recent advances in: Synthetic biology. Libraries of standardized genetic parts which can be used for controlled cellular material production, delivery, and binding; 3D printing. Commercial off-the-shelf components which can be used to make of a pico- to nanoliter cell deposition system; Tissue engineering. Proven cell-compatible support hydrogels and scaffolds can be modified to bind the deposited biomaterials of interest. Objectives: Feasibility and benefit analysis. Two mission contexts span the concept's scope (see below); Proof-of-concept demonstration. A simple grid of two proteins, fluorescent for easy detection, to validate the core technology concept; Proposed implementations for follow-on work. Avenues for future work on each core component (host cell, production control, material delivery, material binding, etc.); Complementary studies exploration. A survey of other emerging areas (in situ resource utilization, protein engineering, etc.) with the potential to multiply our technology's impact. Potential Impacts: This application could dramatically expand manufacturing capabilities on Earth and in space: In situ resource utilization. A far greater range of materials and products will be available from the limited palette offered by in situ resource extraction techniques; Reduced equipment and material upmass for off-Earth habitats. Ready- to-use highly specialized construction materials (radiation hardened, compressive/tensile, light or dense) from an extremely low starting mass; Structured biomaterial production. New ready-to-use macro, micro, and molecular manufacturing techniques for traditional biomaterials such as wood, bone and shell; New and novel biocomposite creation. The ability to create completely novel material composites from any base material that cells can be engineered to produce. Suggested Mission Contexts: ISS part manufacturing. A 'minimal working example' making a finished biomaterial part aboard the International Space Station; A long-term Mars habitat. 'Cradle-to-grave' use at a hypothetical Mars habitat, covering everything from tools to construction materials. Alternate Abstract: Imagine being able to print anything from tools and composite building materials to food and human tissues. Imagine being on Mars with the ability to replace any broken part, whether it's a part of your spacesuit, your habitat, or your own body. We propose a technique that would allow just that. By printing 3D arrays of cells engineered to secrete the necessary materials, the abundant in situ resources of atmosphere and regolith become organic, inorganic, or organic-inorganic composite materials. Such materials include novel, biologically derived materials not previously possible to fabricate.

habitats↗

DOE OSTI2026

The HypA and HypB metallochaperones from Methanococcus maripaludis have unique metal-binding properties and a distinct nickel transfer mechanism

[NiFe] hydrogenases are widespread microbial metalloenzymes that catalyze the reversible conversion of hydrogen (H2) to protons and electrons, playing key roles in energy metabolism. The biosynthesis of the NiFe(CN) 2 CO cofactor involves a suite of maturation proteins, including the HypA and HypB nickel metallochaperones. Here, we define the metal-binding properties, nucleotide-dependent behavior, and functional interplay of HypA and HypB from the hydrogenotrophic methanogenic archaeon, Methanococcus maripaludis . Methanogens have multiple essential nickel-dependent enzymes, so they require efficient systems for nickel delivery that remain largely unexplored. Purified M. maripaludis HypA binds zinc or mononuclear iron at the C-terminal metal binding site, the latter of which has not been reported in other HypA proteins and may serve a unique regulatory role in methanogens. The G-protein metallochaperone HypB binds nickel at the G-domain, which stimulates GTPase activity. Size exclusion chromatography experiments reveal that HypA and HypB form complexes in the presence of nickel, and zinc-bound HypA is optimized for nickel transfer from HypB. The identity of the nucleotide bound to HypB (GDP or GTP) alters the oligomeric state of HypA-HypB complexes, supporting a GTPase-mediated nickel delivery pathway. The HypA-HypB 2 complex configuration is enriched and stable in the presence of GDP and nickel, indicating that this complex delivers nickel to the hydrogenase as opposed to HypA alone. Interestingly, affinity purification-mass spectrometry revealed that HypB interacts with several nickel-dependent proteins, suggesting that HypB may play a broader role in nickel homeostasis in M. maripaludis . Together, this work establishes a biochemical framework for HypAB-mediated nickel trafficking in methanogens.

[NiFe] hydrogenase↗

DOE OSTI2022

An automated liquid jet for fluorescence dosimetry and microsecond radiolytic labeling of proteins

X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method’s overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Crystallization of ApoA1 and ApoE4 Nanolipoprotein Particles and Initial XFEL-Based Structural Studies

Nanolipoprotein particles (NLPs), also called “nanodiscs”, are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via high-density lipoprotein (HDL) and low-density lipoprotein (LDL) maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics, and to develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV’s complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein’s post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Heritable, multinucleotide deletions in plants using viral delivery of a repair exonuclease and guide RNAs

Abstract CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion mutations, which are often too small to disrupt the function of cis-acting regulatory elements. Here, we describe a highly efficient in planta gene editing approach called VirTREX2-HLDel that achieves heritable multinucleotide deletions in both protein-coding genes and noncoding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the 3 prime repair exonuclease 2 (TREX2) and single-guide RNAs. Our method enables recovery of multiplexed heritable deletions and increases the heritable gene editing frequency at poorly edited sites. We identified functional conservation and divergence of MICRORNA164 (miR164) in Nicotiana benthamiana and tomato (Solanum lycopersicum) using VirTREX2-HLDel and observed previously uncharacterized phenotypes in plants with large deletions at this locus. Our viral delivery method reduces the need for tissue culture and will accelerate the understanding of protein-coding and regulatory regions in plants.

59 BASIC BIOLOGICAL SCIENCES↗