Understanding Supramolecular Assembly of Supercharged Proteins.

Ordered supramolecular assemblies have recently been created using electrostatic interactions between oppositely charged proteins. Despite recent progress, the fundamental mechanisms governing the assembly of oppositely supercharged proteins are not fully understood. Here, we use a combination of experiments and computational modeling to systematically study the supramolecular assembly process for a series of oppositely supercharged green fluorescent protein variants. We show that net charge is a sufficient molecular descriptor to predict the interaction fate of oppositely charged proteins under a given set of solution conditions (e.g., ionic strength), but the assembled supramolecular structures critically depend on surface charge distributions. Interestingly, our results show that a large excess of charge is necessary to nucleate assembly and that charged residues not directly involved in interprotein interactions contribute to a substantial fraction (∼30%) of the interaction energy between oppositely charged proteins via long-range electrostatic interactions. Dynamic subunit exchange experiments further show that relatively small, 16-subunit assemblies of oppositely charged proteins have kinetic lifetimes on the order of ∼10-40 min, which is governed by protein composition and solution conditions. Broadly, our results inform how protein supercharging can be used to create different ordered supramolecular assemblies from a single parent protein building block.

Understanding How Coacervates Drive Reversible Small Molecule Reactions to Promote Molecular Complexity.

Liquid-liquid phase-separated coacervate droplets give rise to membraneless compartments that play an important role in the spatial organization and reactivity in cells. Due to their molecularly crowded nature and ability to sequester biomolecules, coacervate droplets create distinct environments for enzymatic reaction kinetics and reaction mechanisms that markedly differ from bulk solution. In this work, we use a combination of experiments and quantitative modeling to understand how coacervate droplets promote reversible small molecule reaction chemistry. In particular, we study a model condensation reaction generating an unstable fluorescent imine in polyacrylic acid-polyethylene glycol coacervate droplets over a range of conditions. At equilibrium, the concentration of the imine product in coacervate droplets is approximately 140-fold larger than that in bulk solution, which arises due to preferential partitioning of reactants and products into coacervate droplets and a reaction equilibrium constant that is roughly threefold larger in coacervate droplets than in solution. A reaction-diffusion model is developed to quantitatively describe how competing reaction and partitioning equilibria govern the spatial distribution of the imine product inside coacervate droplets. Overall, our results show that compartmentalization stabilizes kinetically labile reaction products, which enables larger reactant concentrations in coacervate droplets compared to bulk solution. Broadly, these results provide an improved understanding of how biomolecular condensates promote multistep reaction pathways involving unstable reaction intermediates and suggest how coacervates provide a potential abiotic mechanism to promote molecular complexity.

A critical analysis of electrospray techniques for the determination of accelerated rates and mechanisms of chemical reactions in droplets.

Electrospray and Electrosonic Spray Ionization Mass Spectrometry (ESI-MS and ESSI-MS) have been widely used to report evidence that many chemical reactions in micro- and nano-droplets are dramatically accelerated by factors of ∼102 to 106 relative to macroscale bulk solutions. Despite electrospray's relative simplicity to both generate and detect reaction products in charged droplets using mass spectrometry, substantial complexity exists in how the electrospray process itself impacts the interpretation of the mechanism of these observed accelerated rates. ESI and ESSI are both coupled multi-phase processes, in which analytes in small charged droplets are transferred and detected as gas-phase ions with a mass spectrometer. As such, quantitative examination is needed to evaluate the impact of multiple experimental factors on the magnitude and mechanisms of reaction acceleration. These include: (1) evaporative concentration of reactants as a function of droplet size and initial concentration, (2) competition from gas-phase chemistry and reactions on experimental surfaces, (3) differences in ionization efficiency and ion transmission and (4) droplet charge. We examine (1-4) using numerical models, new ESI/ESSI-MS experimental data, and prior literature to assess the limitations of these approaches and the experimental best practices required to robustly interpret acceleration factors in micro- and nano-droplets produced by ESI and ESSI.

A kinetic description of how interfaces accelerate reactions in micro-compartments.

A kinetic expression is derived to explain how interfaces alter bulk chemical equilibria and accelerate reactions in micro-compartments. This description, aided by the development of a stochastic model, quantitatively predicts previous experimental observations of accelerated imine synthesis in micron-sized emulsions. The expression accounts for how reactant concentration and compartment size together lead to accelerated reaction rates under micro-confinement. These rates do not depend solely on concentration, but rather the fraction of total molecules in the compartment that are at the interface. Although there are ∼107 to 1013 solute molecules in a typical micro-compartment, a kind of "stochasticity" appears when compartment size and reagent concentration yield nearly equal numbers of bulk and interfacial molecules. Although this is distinct from the stochasticity produced by nano-confinement, these results show how interfaces can govern chemical transformations in larger atmospheric, geologic and biological compartments.

Using Nanoparticle X-ray Spectroscopy to Probe the Formation of Reactive Chemical Gradients in Diffusion-Limited Aerosols.

For aerosol particles that exist in highly viscous, diffusion-limited states, steep chemical gradients are expected to form during photochemical aging in the atmosphere. Under these conditions, species at the aerosol surface are more rapidly transformed than molecules residing in the particle interior. To examine the formation and evolution of chemical gradients at aerosol interfaces, the heterogeneous reaction of hydroxyl radicals (OH) on ∼200 nm particles of pure squalane (a branched, liquid hydrocarbon) and octacosane (a linear, solid hydrocarbon) and binary mixtures of the two are used to understand how diffusion limitations and phase separation impact the particle reactivity. Aerosol mass spectrometry is used to measure the effective heterogeneous OH uptake coefficient (γeff) and oxidation kinetics in the bulk, which are compared with the elemental composition of the surface obtained using X-ray photoemission. When diffusion rates are fast relative to the reaction frequency, as is the case for squalane and low-viscosity squalane-octacosane mixtures, the reaction is efficient (γeff ∼ 0.3) and only limited by the arrival of OH to the interface. However, for cases, where the diffusion rates are slower than reaction rates, as in pure octacosane and higher-viscosity squalane-octacosane mixtures, the heterogeneous reaction occurs in a mixing-limited regime and is ∼10× slower (γeff ∼ 0.03). This is in contrast to carbon and oxygen K edge X-ray absorption measurements that show that the octacosane interface is oxidized much more rapidly than that of pure squalane particles. The O/C ratio of the surface (estimated to be the top 6-8 nm of the interface) is measured to change with rate constants of (3.0 ± 0.9) × 10-13 and (8.6 ± 1.2) × 10-13 cm3 molecule-1 s-1 for squalane and octacosane particles, respectively. The differences in surface oxidation rates are analyzed using a previously published reaction-diffusion model, which suggests that a 1-2 nm highly oxidized crust forms on octacosane particles, whereas in pure squalane, the reaction products are homogeneously mixed within the aerosol. This work illustrates how diffusion limitations can form particles with highly oxidized surfaces even at relatively low oxidant exposures, which is in turn expected to influence their microphysics in the atmosphere.

To Be or Not To Be a Molecular Ion: The Role of the Solvent in Photoionization of Arginine.

Application of photoionization mass spectroscopy, a technique capable of assessing protonation states in complex molecules in the gas phase, is challenging for arginine due to its fragility. We report photoionization efficiencies in the valence region of aqueous aerosol particles produced from arginine solutions under various pH and vaporization conditions. By using ab initio calculations, we investigate the stability of different conformers. Our results show that neutral arginine fragments upon ionization in the gas phase but solvation stabilizes the molecular ion, resulting in different photoionization dynamics. We also report the valence-band photoelectron spectra of the aerosol solutions obtained at different pH values.

Studying Chemistry in Micro-compartments by Separating Droplet Generation from Ionization.

Recent studies show that reactions inside micron-sized compartments (e.g., droplets, emulsions) can proceed at significantly accelerated rates and with different mechanisms compared to the same reactions in a macroscopic container. Many of these studies use electrospray ionization (ESI) to both generate droplets and to quantify, via mass spectrometry (MS), droplet reaction kinetics. The highly charged and rapidly evaporating droplets produced in ESI make it difficult to examine precisely the underlying cause for droplet-induced rate enhancements. Additionally, interpretation of the spectra from ESI-MS can be complicated by gas-phase ion-molecule and clustering reactions. Here, we use an approach where droplet generation is separated from ionization, in order to decouple the multiple possible sources of acceleration and to examine more closely the potential role of gas-phase chemistry. The production of sugar phosphates from the reaction of phosphoric acid with simple sugars (a reaction that does not occur in bulk solution but has recently been reported to occur in droplets) is measured using this approach to compare reactivity in droplets (i.e., with compartments) with that in the gas phase (i.e., without compartments). The same product ions that have been previously assigned to in droplet reactions are observed with and without compartmentalization. These results suggest that in some cases, gas-phase processes in the ionization region can potentially complicate the quantification and interpretation of accelerated reactions in droplets using ESI-MS (or one of its variants). In such cases, contributions from in-droplet chemistry cannot be ruled out, but we demonstrate that gas-phase processes can be a significant (and possibly dominant) reaction pathway. We suggest that future studies of rate acceleration in droplets be modified to better assess the potential for non-droplet-related processes. Graphical Abstract ᅟ.

Colliding-Droplet Microreactor: Rapid On-Demand Inertial Mixing and Metal-Catalyzed Aqueous Phase Oxidation Processes.

In-depth investigations of the kinetics of aqueous chemistry occurring in microdroplet environments require experimental techniques that allow a reaction to be initiated at a well-defined point in time and space. Merging microdroplets of different reactants is one such approach. The mixing dynamics of unconfined (airborne) microdroplets have yet to be studied in detail, which is an essential step toward widespread use and application of merged droplet microreactors for monitoring chemical reactions. Here, we present an on-demand experimental approach for initiating chemical reactions in and characterizing the mixing dynamics of colliding airborne microdroplets (40 ± 5 µm diameter) using a streak-based fluorescence microscopy technique. The advantages of this approach include the ability to generate two well-controlled monodisperse microdroplet streams and collide (and thus mix) the microdroplets with high spatial and temporal control while consuming small amounts of sample (<0.1 µL/s). Mixing times are influenced not only by the velocity at which microdroplets collide but also the geometry of the collision (i.e., head-on vs off-center collision). For head-on collisions, we achieve submillisecond mixing times ranging from ∼900 µs at a collision velocity of 0.1 m/s to <200 µs at ∼6 m/s. For low-velocity (<1 m/s) off-center collisions, mixing times were consistent with the head-on cases. For high-velocity (i.e., > 1 m/s) off-center collisions, mixing times increased by as much as a factor of 6 (e.g., at ∼6 m/s, mixing times increased from <200 µs for head-on collisions to ∼1200 µs for highly off-center collisions). At collision velocities >7 m/s, droplet separation and fragmentation occurred, resulting in incomplete mixing. These results suggest a limited range of collision velocities over which complete and rapid mixing can be achieved when using airborne merged microdroplets to, e.g., study reaction kinetics when reaction times are short relative to typical bulk reactor mixing times. We benchmark our reactor using an aqueous-phase oxidation reaction: iron-catalyzed hydroxyl radical production from hydrogen peroxide (Fenton's reaction) and subsequent aqueous-phase oxidation of organic species in solution. Kinetic simulations of our measurements show that quantitative agreement can be obtained using known bulk-phase kinetics for bimolecular reactions in our colliding-droplet microreactor.

Exploring Chemistry in Microcompartments Using Guided Droplet Collisions in a Branched Quadrupole Trap Coupled to a Single Droplet, Paper Spray Mass Spectrometer.

Recent studies suggest that reactions in aqueous microcompartments can occur at significantly different rates than those in the bulk. Most studies have used electrospray to generate a polydisperse source of highly charged microdroplets, leading to multiple confounding factors potentially influencing reaction rates (e.g., evaporation, charge, and size). Thus, the underlying mechanism for the observed enhancement remains unclear. We present a new type of electrodynamic balance-the branched quadrupole trap (BQT)-which can be used to study reactions in microdroplets in a controlled environment. The BQT allows for condensed phase chemical reactions to be initiated by colliding droplets with different reactants and levitating the merged droplet indefinitely. The performance of the BQT is characterized in several ways. Sub-millisecond mixing times as fast as ∼400 µs are measured for low velocity (∼0.1 m/s) collisions of droplets with <40 µm diameters. The reaction of o-phthalaldehyde (OPA) with alanine in the presence of dithiolthreitol is measured using both fluorescence spectroscopy and single droplet paper spray mass spectrometry. The bimolecular rate constant for reaction of alanine with OPA is found to be 84 ± 10 and 67 ± 6 M-1 s-1 in a 30 µm radius droplet and bulk solution, respectively, which demonstrates that bimolecular reaction rate coefficients can be quantified using merged microdroplets and that merged droplets can be used to study rate enhancements due to compartmentalization. Products of the reaction of OPA with alanine are detected in single droplets using paper spray mass spectrometry. We demonstrate that single droplets with <100 pg of analyte can easily be studied using single droplet mass spectrometry.

Low energy electron attenuation lengths in core-shell nanoparticles.

A velocity map imaging spectrometer is used to measure photoemission from free core-shell nanoparticles, where a salt core is coated with a liquid hydrocarbon shell (i.e. squalane). By varying the radial thickness of the hydrocarbon shell, electron attenuation lengths (EALs) are determined by measuring the decay in photoemission intensity from the salt core. In squalane, electrons with kinetic energy (KE) above 2 eV are found to have EALs of 3-5 nm, whereas electrons with smaller KE (<2 eV) have significantly larger EALs of >15 nm. These results (in the context of other energy-resolved EAL measurements) suggest that the energy dependent behavior of low energy electrons is similar in dielectrics when KE > 2 eV. At this energy the EALs do not appear to exhibit strong energy dependence. However, at very low KE (<2 eV), the EALs diverge and appear to be extremely material dependent.

Guanidinium Group Remains Protonated in a Strongly Basic Arginine Solution.

Knowledge of the acid dissociation constant of an amino acid has very important ramifications in the biochemistry of proteins and lipid bilayers in aqueous environments because charge and proton transfer depend on its value. The acid dissociation constant for the guanidinium group in arginine has historically been posited as 12.5, but there is substantial variation in published values over the years. Recent experiments suggest that the dissociation constant for arginine is much higher than 12.5, which explains why the arginine guanidinium group retains its positive charge under all physiological conditions. In this work, we use X-ray photoelectron spectroscopy to study unsupported, aqueous arginine nanoparticles. By varying the pH of the constituent solution, we provide evidence that the guanidinium group is protonated even in a very basic solution. By analyzing the energy shifts in the C and N X-ray photoelectron spectra, we establish a molecular level picture of how charge and proton transport in aqueous solutions of arginine occur.

Probing the Heterogeneous Ozonolysis of Squalene Nanoparticles by Photoemission.

The heterogeneous reaction of ozone (O3) with 200 nm squalene nanoparticles is studied using near-edge X-ray absorption fine structure (NEXAFS) and ultraviolet (UPS) and X-ray photoelectron spectroscopy (XPS). Photoelectrons are detected from free nanoparticle beams using a velocity map imaging (VMI) spectrometer capable of detecting photoelectrons with up to 40 eV of kinetic energy. Heterogeneous kinetics are quantified using changes in the UPS, XPS, and NEXAFS spectrum, yielding uptake coefficients for the decay of the double bonds in squalene of (3.1 ± 0.7) × 10-4, (2.6 ± 0.6) × 10-4, and (2.9 ± 0.7) × 10-4, respectively. When comparing these values with the uptake coefficient, (1.0 ± 0.2) × 10-3, determined by the molecular decay of squalene measured with aerosol mass spectrometry, it is found that on average 1.6 ± 0.2 double bonds are removed for each ozone-squalene reactive collision, suggesting the importance of evaporation of small molecular weight reaction products from the aerosol. From further analysis of the nanoparticle XPS spectrum, it is found that ozonolysis increases the oxygen-to-carbon (O:C) ratio of the aerosol to 0.43 ± 0.03 and produces 16 ± 4% and 84 ± 4% secondary ozonides and carbonyls, respectively. The methods developed here show how aerosol photoemission can be used to quantify heterogeneous reaction on free nanoparticles.

Organosulfate and nitrate formation and reactivity from epoxides derived from 2-methyl-3-buten-2-ol.

Recent work has suggested that 2-methyl-3-butene-2-ol (MBO)-derived epoxide intermediates are responsible for some of the molecular species commonly found in ambient secondary organic aerosol (SOA). Nuclear magnetic resonance techniques were used to study the reaction kinetics and products of two potential MBO-derived epoxides under acidic solution conditions in the presence of sulfate and nitrate nucleophiles. These epoxides were found to undergo reasonably fast acid-catalyzed reaction at typical SOA acidities and to produce a variety of organosulfate and nitrate species. This finding supports a previous supposition that 3-methylbutane-1,2,3-triol and at least some of the MBO-derived organosulfates previously detected on SOA are formed from the reactions of these epoxides. In general, the particular MBO-derived organosulfates and nitrates produced from MBO-derived epoxides and their respective stability toward hydrolysis were similar to those found for isoprene-derived epoxides; the nucleophilic reactions were observed to be quite regiospecific, and the tertiary addition product species were found to hydrolyze on atmospherically relevant time scales.

Rate constants and products of the OH reaction with isoprene-derived epoxides.

Recent laboratory and field work has shown that isoprene-derived epoxides (IEPOX) are crucial intermediates that can explain the existence of a variety of compounds found in ambient secondary organic aerosol (SOA). However, IEPOX species are also able to undergo gas phase oxidation, which competes with the aerosol phase processing of IEPOX. In order to better quantify the atmospheric fate of IEPOX, the gas phase OH reaction rate constants and product formation mechanisms have been determined using a flow tube chemical ionization mass spectrometry technique. The new OH rate constants are generally larger than previous estimations and some features of the product mechanism are well predicted by the Master Chemical Mechanism Version 3.2 (MCM v3.2), while other features are at odds with MCM v3.2. Using a previously proposed kinetic model for the quantitative prediction of the atmospheric fate of IEPOX, it is found that gas phase OH reaction is an even more dominant fate for chemical processing of IEPOX than previously suggested. The present results suggest that aerosol phase processing of IEPOX will be competitive with gas phase OH oxidation only under SOA conditions of high liquid water content and low pH.

Bilateral isolated choroidal melanocytosis.

BACKGROUND: Isolated choroidal melanocytosis refers to patchy melanocytic choroidal hyperpigmentation without the associated scleral or cutaneous pigmentation seen in ocular melanocytosis or oculodermal melanocytosis. Neither bilateral nor diffuse cases, to our knowledge, have been previously described in the literature. METHODS: Case report. PATIENT: A 43-year-old woman without cutaneous or scleral hyperpigmentation or vitiligo was noted to have diffuse patchy melanocytic choroidal hyperpigmentation with feathered margins for nearly 12 clock hours in both eyes. Large choroidal vascular sparing was noted in several areas. RESULTS: B-scan ultrasonography demonstrated a normal choroidal thickness. Cutaneous biopsy with Fontana Masson stain for melanin was within normal limits, without giant melanosomes. CONCLUSIONS: Isolated choroidal melanocytosis may present bilaterally and diffusely.