A nano-catalyst comprised of oxidized Co NPs supported on MgO nano-plates was synthesized via a hydrothermal co-precipitation strategy and calcination in O2 and subsequently in H2 at 250 °C. Spectro-microscopy characterization was performed by scanning transmission electron microscopy, electron energy loss spectroscopy and scanning X-ray transmission microscopy. Surface measurements under H2 and H2 + CO atmospheres were obtained by ambient pressure X-ray photoelectron spectroscopy and in situ X-ray absorption spectroscopy in the 225-480 °C range. These measurements at the atomic and microscopic levels demonstrated that the oxidized Co nanoparticles uniformly decorated the MgO nano-plates. The surfaces are enriched with Co, and with a mixture of Co(OH)2 and CoO under H2 and H2 + CO atmospheres. Under a H2 atmosphere, the outermost surfaces were composed of (lattice) O(2-), CO3(2-) and OH(-). No inorganic carbonates were observed in the bulk. Chemisorbed CO, likely on the oxidized Co surfaces, was observed at the expense of O(2-) under 300 mTorr H2 + CO (2 : 1) at 225 °C. Gas phase CO2 was detected under 32 Torr H2 + CO (2 : 1) at 225 °C upon prolonged reaction time, and was attributed to a surface chemical reaction between O(2-) and chemisorbed CO. Furthermore, sp(3) like carbon species were detected on the otherwise carbon free surface in H2 + CO, which remained on the surface under the subsequent reaction conditions. The formation of sp(3) like hydrocarbons was ascribed to a surface catalytic reaction between the chemisorbed CO and OH(-) as the apparent hydrogen source.

Silica can take many forms, and its interaction with water can change dramatically at the interface. Silica based systems offer a rich tapestry to probe the confinement of water as size and volume can be controlled by various templating strategies and synthetic procedures. To this end, microporous silica nanoparticles have been developed by a reverse microemulsion method utilizing zinc nanoclusters encapsulated in hydroxyl-terminated polyamidoamine (PAMAM-OH) dendrimers as a soft template. These nanoparticles were made tunable within the outer diameter range of 20-50 nm with a core mesopore of 2-15 nm. Synthesized nanoparticles were used to study the effects of surface area and microporous volumes on the vibrational spectroscopy of water. These spectra reveal contributions from bulk interfacial/interparticle water, ice-like surface water, liquid-like water, and hydrated silica surfaces suggesting that microporous silica nanoparticles allow a way to probe silica water interactions at the molecular scale.

The biologically critical, exquisite specificity and efficiency of nucleases, such as those acting in DNA repair and replication, often emerge in the context of multiple other macromolecules. The evolved complexity also makes biologically relevant nuclease assays challenging and low-throughput. Meiotic recombination 11 homolog 1 (MRE11) is an exemplary nuclease that initiates DNA double-strand break (DSB) repair and processes stalled DNA replication forks. Thus, DNA resection by MRE11 nuclease activity is critical for multiple DSB repair pathways as well as in replication. Traditionally, in vitro nuclease activity of purified enzymes is studied either through gel-based assays or fluorescence-based assays like fluorescence resonance energy transfer (FRET). However, adapting these methods for a high-throughput application such as inhibitor screening can be challenging. Gel-based approaches are slow, and FRET assays can suffer from interference and distance limitations. Here we describe an alternative methodology to monitor nuclease activity by measuring the small-angle X-ray scattering (SAXS) interference pattern from gold nanoparticles (Au NPs) conjugated to 5'-ends of dsDNA using X-ray scattering interferometry (XSI). In addition to reporting on the enzyme activity, XSI can provide insight into DNA-protein interactions, aiding in the development of inhibitors that trap enzymes on the DNA substrate. Enabled by efficient access to synchrotron beamlines, sample preparation, and the feasibility of high-throughput XSI data collection and processing pipelines, this method allows for far greater speeds with less sample consumption than conventional SAXS techniques. The reported metrics and methods can be generalized to monitor not only other nucleases but also most other DNA-protein interactions.

Soluble guanylate cyclase (sGC) is the primary receptor for nitric oxide (NO) in mammalian nitric oxide signaling. We determined structures of full-length Manduca sexta sGC in both inactive and active states using cryo-electron microscopy. NO and the sGC-specific stimulator YC-1 induce a 71° rotation of the heme-binding β H-NOX and PAS domains. Repositioning of the β H-NOX domain leads to a straightening of the coiled-coil domains, which, in turn, use the motion to move the catalytic domains into an active conformation. YC-1 binds directly between the β H-NOX domain and the two CC domains. The structural elongation of the particle observed in cryo-EM was corroborated in solution using small angle X-ray scattering (SAXS). These structures delineate the endpoints of the allosteric transition responsible for the major cyclic GMP-dependent physiological effects of NO.

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NP^NTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NP^NTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NP^NTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

Human serum albumin (HSA) constitutes the primary transporter of fatty acids, bilirubin, and other plasma compounds. The binding, transport, and release of its cargos strongly depend on albumin conformation, which is affected by bound ligands induced by physiological and pathological conditions. HSA is both highly oxidized and heavily loaded with fatty acids and bilirubin in chronic liver disease. By employing small-angle X-ray scattering we show that HSA from the plasma of chronic liver disease patients undergoes a distinct opening compared to healthy donors. The extent of HSA opening correlates with clinically relevant variables, such as the model of end-stage liver disease score, bilirubin, and fatty acid levels. Although the mild oxidation of HSA in vitro does not alter overall structure, the alteration of patients' HSA correlates with its redox state. This study connects clinical data with structural visualization of albumin dynamicity in solution and underlines the functional importance of albumin's inherent flexibility.

Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand breaks (DSBs) forms DNA-PK holoenzyme as a critical initiating step for non-homologous end joining (NHEJ) repair of DSBs produced by radiation and chemotherapies. Advanced cryo-electron microscopy (cryo-EM) imaging together with breakthrough macromolecular X-ray crystal (MX) structures of KU and DNA-PKcs recently enabled visualization of the ∼600 kDa DNA-PK assembly at near atomic resolution. These important static structures provide the foundation for definition and interpretation of functional movements crucial to mechanistic understanding that can be tested through solution state structure analysis. We herein therefore leverage Cryo-EM and MX structures for the interpretation of synchrotron small-angle X-ray scattering (SAXS) data on DNA-PK conformations in solution to inform the structural mechanism for NHEJ initiation. SAXS, which measures thermodynamic solution-state conformational states and assemblies outside of cryo- and solid-state conditions, unveils the inherent flexibility of KU, DNA-PKcs and DNA-PK. The combined structural measurements reveal mobility of KU80 C-terminal region (KU80CTR), motion/plasticity of HEAT (DNA-PKcs Huntingtin, Elongation Factor 3, PP2 A, and TOR1) regions, allosteric switching upon DNA-PKcs autophosphorylation, and dimeric arrangements of DNA-PK assembly. Importantly, the results uncover displacement of the N-terminal HEAT domain during autophosphorylation as suitable for a regulated release mechanism of DNA-PKcs from DNA-PK to control unproductive access to toxic and mutagenic DNA repair intermediates. These integrated analyses show that the marriage of SAXS with cryo-EM leverages the strengths of both techniques to enable assessment of functional conformations and flexibility defining atomic-resolution molecular mechanisms for DSB repair.

When nanoparticles enter biological environments, proteins adsorb to form the "protein corona" which alters nanoparticle biodistribution and toxicity. Herein, we measure protein corona formation on DNA-functionalized single-walled carbon nanotubes (ssDNA-SWCNTs), a nanoparticle used widely for sensing and delivery, in blood plasma and cerebrospinal fluid. We characterize corona composition by mass spectrometry, revealing high-abundance corona proteins involved in lipid binding, complement activation, and coagulation. We investigate roles of electrostatic and entropic interactions driving selective corona formation. Lastly, we study real-time protein binding on ssDNA-SWCNTs, obtaining agreement between enriched proteins binding strongly and depleted proteins binding marginally, while highlighting cooperative adsorption mechanisms. Knowledge of protein corona composition, formation mechanisms, and dynamics informs nanoparticle translation from in vitro design to in vivo application.