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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Viral delivery of recombinases activates heritable genetic switches in plants

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents; however, they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from ~0.6kb to ~1.5kb and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue and led to heritable modifications at high frequency. Here, an excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery and offer insights into infection dynamics that may be useful in developing future viral vectors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Serial crystallography using automated drop dispensing

Automated, pulsed liquid-phase sample delivery has the potential to greatly improve the efficiency of both sample and photon use at pulsed X-ray facilities. In this work, an automated drop on demand (DOD) system that accelerates sample exchange for serial femtosecond crystallography (SFX) is demonstrated. Four different protein crystal slurries were tested, and this technique is further improved here with an automatic sample-cycling system whose effectiveness was verified by the indexing results. Here, high-throughput SFX screening is shown to be possible at free-electron laser facilities with very low risk of cross contamination and minimal downtime. The development of this technique will significantly reduce sample consumption and enable structure determination of proteins that are difficult to crystallize in large quantities. This work also lays the foundation for automating sample delivery.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗
DOE OSTI2025

A nut-and-bolt assembly of the bimodular large progenitor botulinum neurotoxin complex

Botulinum neurotoxin serotype A (BoNT/A) is naturally produced by bacteria along with four nontoxic neurotoxin-associated proteins (NTNH, HA70, HA33, and HA17), forming a bimodular large progenitor toxin complex (L-PTC). The BoNT/A–NTNH complex protects the toxin from adverse environment, while the complex consisting of HA proteins facilitates toxin absorption during oral intoxication. How these two independent modules assemble into the L-PTC remains unclear. Here, we report the crystal structure of the BoNT/A–NTNH–HA70 complex at ~2.9-Å resolution. The structure reveals that the BoNT/A–NTNH complex is anchored into a concentric double β-barrel channel of trimeric HA70 through a short β-hairpin of NTNH (termed nLoop), resembling a nut-and-bolt attachment. We find that the nLoop of NTNH is strictly conserved across HA-containing BoNT complexes and that NTNH–HA70 binding is interchangeable among them. Furthermore, we demonstrate that the nLoop functions as a minimal motif enabling attachment of a protein-of-interest to the HA complex, with potential applications in oral biologics delivery.

Science & Technology - Other Topics↗
DOE OSTI2023

A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H 2 formation

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe 7 S 9 MoC-homocitrate) as a critical N 2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E 4 (4H), which has accumulated 4[e — /H + ] as two bridging hydrides, Fe2–H–Fe6 and Fe3–H–Fe7, and protons bound to two sulfurs. E 4 (4H) is poised to bind/reduce N 2 as driven by mechanistically-coupled H 2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H 2 as the enzyme relaxes to state E 2 (2H), containing 2[e — /H + ] as a hydride and sulfur-bound proton; accumulation of E 4 (4H) in α-V70I is enhanced by HP suppression. EPR and 95 Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e — /H + ] to the E 0 state of the WT MoFe protein and to both α-V70I conformations generating E 2 (2H) that contains the Fe3–H–Fe7 bridging hydride; accumulation of another 2[e — /H + ] generates E 4 (4H) with Fe2–H–Fe6 as the second hydride. E 4 (4H) in WT enzyme and a minority α-V70I E 4 (4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2–H–Fe6 followed by slower HP of Fe3–H–Fe7, which leads to transient accumulation of E 2 (2H) containing Fe3–H–Fe7. In the dominant α-V70I E 4 (4H) conformation, HP of Fe2–H–Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3–H–Fe7 occurs first and the resulting E 2 (2H) contains Fe2–H–Fe6. Importantly, it is this HP suppression in E 4 (4H) that enables α-V70I MoFe to accumulate E 4 (4H) in high occupancy. In addition, HP suppression in α-V70I E 4 (4H) kinetically unmasks hydride reductive-elimination without N 2 -binding, a process that is precluded in WT enzyme.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Predicting transport of intra-articularly injected growth factor fusion proteins into human knee joint cartilage

There are no drugs or treatment methods known to prevent the development of post-traumatic osteoarthritis (PTOA), a type of osteoarthritis (OA) that is triggered by traumatic joint injuries and accounts for ~12% of the nearly 600 million OA cases worldwide. Lack of effective drug delivery techniques remains a major challenge in developing clinically effective treatments, but cationic delivery carriers can help overcome this challenge. Scaling up treatments that are effective in in vitro models to achieve success in preclinical in vivo models and clinical trials is also a challenging problem in the field. Here we use a cationic green fluorescent protein (GFP) as a carrier to deliver Insulin-Like Growth Factor 1 (IGF-1), a drug considered as a potential therapeutic for PTOA. GFP-IGF-1 conjugates were first synthesized as fusion proteins with different polypeptide linkers, and their transport properties were characterized in human cartilage explants. In vitro experimental data were used to develop a predictive mathematical transport model that was validated using an independent in vitro experimental data set. In this study, the model was used to predict the transport of these fusion proteins upon intra-articular injection into human knee joints. The predictions included results for the rate and extent of fusion protein penetration into cartilage, and the maximum levels of fusion proteins that would escape into systemic circulation through the joint capsule. Together, our transport measurements and model set the stage for translation of such explant culture studies to in vivo preclinical studies and potentially clinical application.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology

Abstract Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana . Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

Occhialini, Alessandro (ORCID:000000021162798X)↗
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↗
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↗
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↗