Coiled-Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability.

The organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Furthermore, the stabilization of enzyme activity has many practical applications in biotechnology and medicine. Here we describe the symmetry-directed design of an extremely stable, enzymatically active, hollow protein cage of Mr ≈ 2.1 MDa with dimensions similar to those of a small icosahedral virus. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain, and cryo-EM and by atomic force and IR-photoinduced force microscopy established that Ico8 assembles into a flexible hollow cage comprising 20 copies of the esterase trimer, 60 protein subunits in total, with overall icosahedral geometry. Notably, the cages formed by Ico8 proved to be extremely stable toward thermal and chemical denaturation: whereas TriEst was unfolded by heating ( Tm ≈ 75 °C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 °C or in 8 M guanidine hydrochloride. The increased stability of the cages is a new property that emerges from the higher-order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.

Bouncier Particles at Night: Biogenic Secondary Organic Aerosol Chemistry and Sulfate Drive Diel Variations in the Aerosol Phase in a Mixed Forest.

Aerosol phase state is critical for quantifying aerosol effects on climate and air quality. However, significant challenges remain in our ability to predict and quantify phase state during its evolution in the atmosphere. Herein, we demonstrate that aerosol phase (liquid, semisolid, solid) exhibits a diel cycle in a mixed forest environment, oscillating between a viscous, semisolid phase state at night and liquid phase state with phase separation during the day. The viscous nighttime particles existed despite higher relative humidity and were independently confirmed by bounce factor measurements and atomic force microscopy. High-resolution mass spectrometry shows the more viscous phase state at night is impacted by the formation of terpene-derived and higher molecular weight secondary organic aerosol (SOA) and smaller inorganic sulfate mass fractions. Larger daytime particulate sulfate mass fractions, as well as a predominance of lower molecular weight isoprene-derived SOA, lead to the liquid state of the daytime particles and phase separation after greater uptake of liquid water, despite the lower daytime relative humidity. The observed diel cycle of aerosol phase should provoke rethinking of the SOA atmospheric lifecycle, as it suggests diurnal variability in gas-particle partitioning and mixing time scales, which influence aerosol multiphase chemistry, lifetime, and climate impacts.

Isoprene-Derived Organosulfates: Vibrational Mode Analysis by Raman Spectroscopy, Acidity-Dependent Spectral Modes, and Observation in Individual Atmospheric Particles.

Isoprene, the most abundant biogenic volatile organic compound (BVOC) in the atmosphere, and its low-volatility oxidation products lead to secondary organic aerosol (SOA) formation. Isoprene-derived organosulfates formed from reactions of isoprene oxidation products with sulfate in the particle phase are a significant component of SOA and can hydrolyze forming polyols. Despite characterization by mass spectrometry, their basic structural and spectroscopic properties remain poorly understood. Herein, Raman microspectroscopy and density functional theory (DFT) calculations (CAM-B3LYP level of theory) were combined to analyze the vibrational modes of key organosulfates, 3-methyltetrol sulfate esters (racemic mixture of two isomers), and racemic 2-methylglyceric acid sulfate ester, and hydrolysis products, 2-methyltetrols, and 2-methylglyceric acid. Two intense vibrational modes were identified, ν(RO-SO3) (846 ± 4 cm-1) and νs(SO3) (1065 ± 2 cm-1), along with a lower intensity δ(SO3) mode (586 ± 2 cm-1). For 2-methylglyceric acid and its sulfate esters, deprotonation of the carboxylic acid at pH values above the pKa decreased the carbonyl stretch frequency (1724 cm-1), while carboxylate modes grew in for νs(COO-) and νa(COO-) at 1413 and 1594 cm-1, respectively. The ν(RO-SO3) and νs(SO3) modes were observed in individual atmospheric particles and can be used in future studies of complex SOA mixtures to distinguish organosulfates from inorganic sulfate or hydrolysis products.

Atomic Force Microscopy-Infrared Spectroscopy of Individual Atmospheric Aerosol Particles: Subdiffraction Limit Vibrational Spectroscopy and Morphological Analysis.

Chemical analysis of atmospheric aerosols is an analytical challenge, as aerosol particles are complex chemical mixtures that can contain hundreds to thousands of species in attoliter volumes at the most abundant sizes in the atmosphere (∼100 nm). These particles have global impacts on climate and health, but there are few methods available that combine imaging and the detailed molecular information from vibrational spectroscopy for individual particles <500 nm. Herein, we show the first application of atomic force microscopy with infrared spectroscopy (AFM-IR) to detect trace organic and inorganic species and probe intraparticle chemical variation in individual particles down to 150 nm. By detecting photothermal expansion at frequencies where particle species absorb IR photons from a tunable laser, AFM-IR can study particles smaller than the optical diffraction limit. Combining strengths of AFM (ambient pressure, height, morphology, and phase measurements) with photothermal IR spectroscopy, the potential of AFM-IR is shown for a diverse set of single-component particles, liquid-liquid phase separated particles (core-shell morphology), and ambient atmospheric particles. The spectra from atmospheric model systems (ammonium sulfate, sodium nitrate, succinic acid, and sucrose) had clearly identifiable features that correlate with absorption frequencies for infrared-active modes. Additionally, molecular information was obtained with <100 nm spatial resolution for phase separated particles with a ∼150 nm shell and 300 nm core. The subdiffraction limit capability of AFM-IR has the potential to advance understanding of particle impacts on climate and health by improving analytical capabilities to study water uptake, heterogeneous reactivity, and viscosity.

Inland Sea Spray Aerosol Transport and Incomplete Chloride Depletion: Varying Degrees of Reactive Processing Observed during SOAS.

Multiphase reactions involving sea spray aerosol (SSA) impact trace gas budgets in coastal regions by acting as a reservoir for oxidized nitrogen and sulfur species, as well as being a source of halogen gases (HCl, ClNO2, etc.). Whereas most studies of multiphase reactions on SSA have focused on marine environments, far less is known about SSA transported inland. Herein, single-particle measurements of SSA are reported at a site >320 km from the Gulf of Mexico, with transport times of 7-68 h. Samples were collected during the Southern Oxidant and Aerosol Study (SOAS) in June-July 2013 near Centreville, Alabama. SSA was observed in 93% of 42 time periods analyzed. During two marine air mass periods, SSA represented significant number fractions of particles in the accumulation (0.2-1.0 µm, 11%) and coarse (1.0-10.0 µm, 35%) modes. Chloride content of SSA particles ranged from full to partial depletion, with 24% of SSA particles containing chloride (mole fraction of Cl/Na ≥ 0.1, 90% chloride depletion). Both the frequent observation of SSA at an inland site and the range of chloride depletion observed suggest that SSA may represent an underappreciated inland sink for NOx/SO2 oxidation products and a source of halogen gases.

Direct Measurement of pH in Individual Particles via Raman Microspectroscopy and Variation in Acidity with Relative Humidity.

Atmospheric aerosol acidity is an important characteristic of aqueous particles, which has been linked to the formation of secondary organic aerosol by catalyzing reactions of oxidized organic compounds that have partitioned to the particle phase. However, aerosol acidity is difficult to measure and traditionally estimated using indirect methods or assumptions based on composition. Ongoing disagreements between experiments and thermodynamic models of particle acidity necessitate improved fundamental understanding of pH and ion behavior in high ionic strength atmospheric particles. Herein, Raman microspectroscopy was used to determine the pH of individual particles (H2SO4+MgSO4) based on sulfate and bisulfate concentrations determined from νs(SO4(2-)) and νs(HSO4(-)), the acid dissociation constant, and activity coefficients from extended Debye-Hückel calculations. Shifts in pH and peak positions of νs(SO4(2-)) and νs(HSO4(-)) were observed as a function of relative humidity. These results indicate the potential for direct spectroscopic determination of pH in individual particles and the need to improve fundamental understanding of ion behavior in atmospheric particles.

Surface Enhanced Raman Spectroscopy Enables Observations of Previously Undetectable Secondary Organic Aerosol Components at the Individual Particle Level.

The first use of surface enhanced Raman spectroscopy (SERS) to detect trace organic and/or inorganic species in ambient atmospheric aerosol particles is presented. This new analytical method provides direct, spectroscopic detection of species present at attogram to femtogram levels in individual submicrometer atmospheric particles. An array of spectral features resulting from organic functional groups in secondary organic aerosol (SOA) material were observed in individual particles impacted on silver nanoparticle-coated substrates. The results demonstrate the complexity of organic and inorganic species in SOA formed by oxidation of biogenic volatile organic compounds (BVOCs) at the single particle level. While SOA composition is frequently assumed to be homogeneous between and within individual particles, substantial particle-to-particle variability in SOA composition and changes on scales <1 µm were observed. The observations obtained with this new method demonstrate the power of SERS to probe difficult to detect inter- and intraparticle variability in ambient SOA particles.

An In Situ Method for Sizing Insoluble Residues in Precipitation and Other Aqueous Samples.

Particles are frequently incorporated into clouds or precipitation, influencing climate by acting as cloud condensation or ice nuclei, taking up coatings during cloud processing, and removing species through wet deposition. Many of these particles, particularly ice nuclei, can remain suspended within cloud droplets/crystals as insoluble residues. While previous studies have measured the soluble or bulk mass of species within clouds and precipitation, no studies to date have determined the number concentration and size distribution of insoluble residues in precipitation or cloud water using in situ methods. Herein, for the first time we demonstrate that Nanoparticle Tracking Analysis (NTA) is a powerful in situ method for determining the total number concentration, number size distribution, and surface area distribution of insoluble residues in precipitation, both of rain and melted snow. The method uses 500 µL or less of liquid sample and does not require sample modification. Number concentrations for the insoluble residues in aqueous precipitation samples ranged from 2.0-3.0(±0.3)×108 particles cm-3, while surface area ranged from 1.8(±0.7)-3.2(±1.0)×107 µm2 cm-3. Number size distributions peaked between 133-150 nm, with both single and multi-modal character, while surface area distributions peaked between 173-270 nm. Comparison with electron microscopy of particles up to 10 µm show that, by number, > 97% residues are <1 µm in diameter, the upper limit of the NTA. The range of concentration and distribution properties indicates that insoluble residue properties vary with ambient aerosol concentrations, cloud microphysics, and meteorological dynamics. NTA has great potential for studying the role that insoluble residues play in critical atmospheric processes.

Rapid Kinetics of Size and pH-Dependent Dissolution and Aggregation of Silver Nanoparticles in Simulated Gastric Fluid.

As silver nanoparticles (AgNPs) are used in a wide array of commercial products and can enter the human body through oral exposure, it is important to understand the fundamental physical and chemical processes leading to changes in nanoparticle size under the conditions of the gastrointestinal (GI) tract. Rapid AgNP growth was observed using nanoparticle tracking analysis with 30 s resolution over a period of 17 min in simulated gastric fluid (SGF) to explore rapid kinetics as a function of pH (SGF at pH 2, 3.5, 4.5 and 5), size (20 and 110 nm AgNPs), and nanoparticle coating (citrate and PVP). Growth was observed for 20 nm AgNP at each pH, decreasing in rate with increasing pH, with the kinetics shifting from second-order to first-order. The 110 nm AgNP showed growth at ≤3.5 pH, with no growth observed at higher pH. This behavior can be explained by the generation of Ag+ in acidic environments, which precipitates with Cl-, leading to particle growth and facilitating particle aggregation by decreasing their electrostatic repulsion in solution. These results highlight the need to further understand the importance of initial size, physicochemical properties, and kinetics of AgNPs after ingestion to assess potential toxicity.