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At least 415 records · Page 23

Electron and ion spectroscopy of azobenzene in the valence and core shells

Azobenzene is a prototype and a building block of a class of molecules of extreme technological interest as molecular photo-switches. We present a joint experimental and theoretical study of its response to irradiation with light across the UV to x-ray spectrum. The study of valence and inner shell photo-ionization and excitation processes combined with measurement of valence photoelectron-photoion coincidence and mass spectra across the core thresholds provides a detailed insight into the site- and state-selected photo-induced processes. Photo-ionization and excitation measurements are interpreted via the multi-configurational restricted active space self-consistent field method corrected by second order perturbation theory. Using static modeling, we demonstrate that the carbon and nitrogen K edges of azobenzene are suitable candidates for exploring its photoinduced dynamics thanks to the transient signals appearing in background-free regions of the NEXAFS and XPS.

Chemistry↗

Osteocalcin binds to a GPRC6A Venus fly trap allosteric site to positively modulate GPRC6A signaling

GPRC6A, a member of the Family C G-protein coupled receptors, regulates energy metabolism and sex hormone production and is activated by diverse ligands, including cations, L-amino acids, the osteocalcin (Ocn) peptide and the steroid hormone testosterone. We sought a structural framework for the ability of multiple distinct classes of ligands to active GPRC6A. We created a structural model of GPRC6A using Alphafold2. Using this model we explored a putative orthosteric ligand binding site in the bilobed Venus fly trap (VFT) domain of GPRC6A and two positive allosteric modulator (PAM) sites, one in the VFT and the other in the 7 transmembrane (7TM) domain. We provide evidence that Ocn peptides act as a PAM for GPRC6A by binding to a site in the VFT that is distinct from the orthosteric site for calcium and L-amino acids. In agreement with this prediction, alternatively spliced GPRC6A isoforms 2 and 3, which lack regions of the VFT, and mutations in the computationally predicted Ocn binding site, K352E and H355P, prevent Ocn activation of GPRC6A. These observations explain how dissimilar ligands activate GPRC6A and set the stage to develop novel molecules to activate and inhibit this previously poorly understood receptor.

59 BASIC BIOLOGICAL SCIENCES↗

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP–importin α interactions

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin β and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Å crystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2–ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117–196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Leveraging Topological Events in Tracking Graphs for Understanding Particle Diffusion

Abstract Single particle tracking (SPT) of fluorescent molecules provides significant insights into the diffusion and relative motion of tagged proteins and other structures of interest in biology. However, despite the latest advances in high‐resolution microscopy, individual particles are typically not distinguished from clusters of particles. This lack of resolution obscures potential evidence for how merging and splitting of particles affect their diffusion and any implications on the biological environment. The particle tracks are typically decomposed into individual segments at observed merge and split events, and analysis is performed without knowing the true count of particles in the resulting segments. Here, we address the challenges in analyzing particle tracks in the context of cancer biology. In particular, we study the tracks of KRAS protein, which is implicated in nearly 20% of all human cancers, and whose clustering and aggregation have been linked to the signaling pathway leading to uncontrolled cell growth. We present a new analysis approach for particle tracks by representing them as tracking graphs and using topological events – merging and splitting, to disambiguate the tracks. Using this analysis, we infer a lower bound on the count of particles as they cluster and create conditional distributions of diffusion speeds before and after merge and split events. Using thousands of time‐steps of simulated and in‐vitro SPT data, we demonstrate the efficacy of our method, as it offers the biologists a new, detailed look into the relationship between KRAS clustering and diffusion speeds.

McDonald, T.↗

Prospects for detecting UF6 hydrolysis intermediates by mass spectrometry, resonance Raman, and NQR spectroscopy

Simulations are performed to consider several spectrometric and spectroscopic candidates for elucidating the mechanism of the hydrolysis of uranium hexafluoride (UF6). This study is among the first to benchmark the def-mTZVP basis sets for actinide-containing molecules, and it is shown to be a suitable basis set for surveying geometrical structures and vibrational spectra when used in conjunction with density functional theory. An experiment is proposed coupling mass spectrometric ion selection with vibrational spectroscopy, and supporting infrared and Raman spectral simulations demonstrate that ionization blue-shifts bands and can change their qualitative features. Ultraviolet resonance Raman is shown to have good prospects for discriminating U–O–U bridged intermediates, evidence for which was observed recently [L. E. McNamara et al., J. Phys. Chem. A 130, 775–786 (2026)]. As a more speculative approach, we also consider the prospect of addressing 233U or 235U nuclei by quadrupole resonance (NQR) spectroscopy for assigning early stage intermediate complexes. In doing so, we provide a first order-of-magnitude estimate for the collision-induced NQR signal for the UF6 dimer, which is of interest for the interpretation of a fast decoherence time observed in liquid-phase nuclear magnetic resonance. Having provided critical insights into the formation and stability of UF6 hydrolysis intermediates in previous studies, this computational spectroscopy survey is expected to help guide and expedite future laboratory campaigns.

Lutz, Jesse J. [Center for Computing Research, San↗

Laser-Plasma Spectroscopy of Hydroxyl with Applications

This article discusses laser-induced laboratory-air plasma measurements and analysis of hydroxyl (OH) ultraviolet spectra. The computations of the OH spectra utilize line strength data that were developed previously and that are now communicated for the first time. The line strengths have been utilized extensively in interpretation of recorded molecular emission spectra and have been well-tested in laser-induced fluorescence applications for the purpose of temperature inferences from recorded data. Moreover, new experiments with Q-switched laser pulses illustrate occurrence of molecular recombination spectra for time delays of the order of several dozen of microseconds after plasma initiation. The OH signals occur due to the natural humidity in laboratory air. Centrifugal stretching of the Franck-Condon factors and r-centroids are included in the process of determining the line strengths that are communicated as a Supplementary File. Laser spectroscopy applications of detailed OH computations include laser-induced plasma and combustion analyses, to name but two applications. This work also includes literature references that address various diagnosis applications.

47 OTHER INSTRUMENTATION↗

Device and Method for Parallel Measurement of Phosphoproteome and Proteome from Single Cells

We present the development of an immobilized metal affinity chromatography (IMAC) chip designed to enable nanoscale phosphopeptide enrichment within microfabricated nanowells. This novel platform leverages surface chemistry to immobilize high-density Nickel-Nitrilotriacetic Acid (Ni-NTA) molecules on nanowells, followed by applying Fe 3+ . The nanowell surface serves as a capture media to enrich phosphopeptides based on IMAC. The system's efficiency was validated using ß-casein as a model protein, demonstrating the chip’s capability to significantly enrich phosphopeptides. Future applications of this technology are anticipated to enable the detection of over 100 phosphopeptides from individual cells and more than 500 phosphopeptides from pools of 100 cells, offering exciting potential for single-cell phosphoproteomics. We will next apply an integrated proteomics workflow to perform multi-omics measurements, including single-cell isolation, protein digestion, and phosphopeptide enrichment, followed by LC-MS analysis of both the global proteome and phosphoproteome. Future research will explore the use of this technology to study phosphorylation dynamics in cancer cells, enhancing our understanding of cellular signaling and disease mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗

Resonant Auger decay of dissociating CH 3 I near the I 4d threshold

Resonant Auger processes provide a unique perspective on electronic interactions and excited vibrational and electronic states of molecular ions. Here, new data are presented on the resonant Auger decay of excited CH 3 I in the region just below the I 4d -1 ionization threshold. The resonances include the Rydberg series converging to the five spin–orbit and ligand-field split CH 3 I (I 4d -1 ) thresholds, as well as resonances corresponding to excitation from the I 4d 5/2,3/2 orbitals into the σ* lowest unoccupied molecular orbital. This study focuses on participator decay that populates the lowest lying states of CH 3 I + , in particular, the X̃ 2 E 3/2 and 2 E 1/2 states, and on spectator decay that populates the lowest-lying (CH 3 I 2+ )σ* states of CH 3 I + . The CH 3 I (I 4d -1 )σ* resonances are broad, and dissociation to CH 3 + I competes with the autoionization of the core-excited states. Auger decay as the molecule dissociates produces a photoelectron spectrum with a long progression (up to v 3 + ~ 25) in the C–I stretching mode of the X̃ 2 E 3/2 and 2 E 1/2 states, providing insights into the shape of the dissociative core-excited surface. Further, the observed spectator decay processes indicate that CH 3 I + is formed on the repulsive wall of the lower-lying (CH 3 I 2+ )σ* potentials, and the photon-energy dependence of the processes provides insights into the relative slopes of the (4d -1 )σ* and (CH 3 I 2+ )σ* potential surfaces. Data are also presented for the spectator decay of higher lying CH 3 I (I 4d -1 )nl Rydberg resonances. Photoelectron angular distributions for the resonant Auger processes provide additional information that helps distinguish these processes from the direct ionization signal.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Thermodynamics of calcium binding to heparin: Implications of solvation and water structuring for polysaccharide biofunctions

Heparan sulfates are found in all animal tissues and have essential roles in living systems. This family of biomacromolecules modulates binding to calcium ions (Ca 2+ ) in low free energy reactions that influence biochemical processes from cell signaling and anticoagulant efficacy to biomineralization. Despite their ubiquity, the thermodynamic basis for how heparans and similarly functionalized biomolecules regulate Ca 2+ interactions is not yet established. Using heparosan (Control) and heparins with different positions of sulfate groups, we quantify how SO 3 − and COO − content and SO 3 − position modulate Ca 2+ binding by isothermal titration calorimetry. The free energy of all heparin-Ca2+ interactions (ΔG rxn ) is dominated by entropic contributions due to favorable water release from polar, hydrophilic groups. Heparin with both sulfate esters (O-SO 3 − ) and sulfamides (N-SO 3 − ) has the strongest binding to Ca 2+ compared to heparosan and to heparin with only O-SO 3 − groups (~3X). By linking Ca 2+ binding thermodynamics to measurements of the interfacial energy for calcite (CaCO 3 ) crystallization onto polysaccharides, we show molecule-specific differences in nucleation rate can be explained by differences in water structuring during Ca 2+ interactions. A large entropic term (-TΔS rxn ) upon Ca 2+ –polysaccharide binding correlates with high interfacial energy to CaCO 3 nucleation. Combining our measurements with literature values indicates many Ca 2+ –polysaccharide interactions have a shared thermodynamic signature. The resulting enthalpy–entropy compensation relationship suggests these interactions are generally dominated by water restructuring involving few conformational changes, distinct from Ca 2+ –protein binding. Our findings quantify the thermodynamic origins of heparin-specific interactions with Ca 2+ and demonstrate the contributions of solvation and functional group position during biomacromolecule-mediated ion regulation.

15 GEOTHERMAL ENERGY↗

A detailed experimental and modeling comparison of molecular radiative heat loss in a spark-ignition engine

Radiative heat transfer has been chiefly considered negligible in internal combustion engines, except for Diesel engines where soot radiation was recognized as a significant radiative transfer source. Only more recently, detailed simulations have shown that molecular radiation can be substantial as well. In extension to this, molecular radiative heat transfer can reach detectable levels of about 5–10% of the total heat transfer in spark-ignited engines. The broadband radiative nature of the significant emitting molecules, carbon dioxide and water, makes it necessary to address whether radiative trapping plays a substantial role in either total heat loss or energy redistribution within the cylinder. An experimental setup that allows measurements of the infrared emissions at a 2-crank-angle-degree resolution was developed to determine the importance of radiative trapping by carbon dioxide and water. Measurements were conducted in the well-characterized and documented TCC-III engine at the University of Michigan. The engine operated on a stoichiometric propane/air mixture at a speed of 1,300 RPM. Large Eddy Simulations with added line-by-line photon Monte-Carlo simulations of the molecular radiation were an integral part of this study to plan and devise the measurements. Post-processing of the simulation data included an accurate representation of the experimental volume from where infrared signals are collected. Additionally, the experimental data were used for validation of the photon Monte-Carlo simulations. Joint analysis of experimental and simulated spectra allowed quantifying the significance of radiative trapping. Results show the significant role of radiative trapping when predicting radiative heat transfer in the TCC-III engine.

42 ENGINEERING↗

Toward a quantitative description of solvation structure: a framework for differential solution scattering measurements

Appreciating that the role of the solute–solvent and other outer-sphere interactions is essential for understanding chemistry and chemical dynamics in solution, experimental approaches are needed to address the structural consequences of these interactions, complementing condensed-matter simulations and coarse-grained theories. High-energy X-ray scattering (HEXS) combined with pair distribution function analysis presents the opportunity to probe these structures directly and to develop quantitative, atomistic models of molecular systems in situ in the solution phase. However, at concentrations relevant to solution-phase chemistry, the total scattering signal is dominated by the bulk solvent, prompting researchers to adopt a differential approach to eliminate this unwanted background. Though similar approaches are well established in quantitative structural studies of macromolecules in solution by small- and wide-angle X-ray scattering (SAXS/WAXS), analogous studies in the HEXS regime—where sub-ångström spatial resolution is achieved—remain underdeveloped, in part due to the lack of a rigorous theoretical description of the experiment. To address this, herein we develop a framework for differential solution scattering experiments conducted at high energies, which includes concepts of the solvent-excluded volume introduced to describe SAXS/WAXS data, as well as concepts from the time-resolved X-ray scattering community. Our theory is supported by numerical simulations and experiment and paves the way for establishing quantitative methods to determine the atomic structures of small molecules in solution with resolution approaching that of crystallography.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Fast event-based electron counting for small-molecule structure determination by MicroED

Electron counting helped realize the resolution revolution in single-particle cryoEM and is now accelerating the determination of MicroED structures. Its advantages are best demonstrated by new direct electron detectors capable of fast (kilohertz) event-based electron counting (EBEC). This strategy minimizes the inaccuracies introduced by coincidence loss (CL) and promises rapid determination of accurate structures. We used the Direct Electron Apollo camera to leverage EBEC technology for MicroED data collection. Given its ability to count single electrons, the Apollo collects high-quality MicroED data from organic small-molecule crystals illuminated with incident electron beam flux densities as low as 0.01–0.045 e − /Å 2 /s. Under even the lowest flux density (0.01 e − /Å 2 /s) condition, fast EBEC data produced ab initio structures of a salen ligand (268 Da) and biotin (244 Da). Each structure was determined from a 100° wedge of data collected from a single crystal in as few as 50 s, with a delivered fluence of only ∼0.5 e − /Å 2 . Fast EBEC data collected with a fluence of 2.25 or 3.33 e − /Å 2 also facilitated a 1.5 Å structure of thiostrepton (1665 Da). While refinement of these structures appeared unaffected by CL, a CL adjustment applied to EBEC data further improved the distribution of intensities measured from the salen ligand and biotin crystals. However, CL adjustment only marginally improved the refinement of their corresponding structures, signaling the already high counting accuracy of detectors with counting rates in the kilohertz range. Overall, by delivering low-dose structure-worthy data, fast EBEC collection strategies open new possibilities for high-throughput MicroED.

EBEC↗

Enabling Ultralow Volume Analysis with a High-Resolution Ion Mobility Mass Spectrometry Platform

Of all the molecules thought to exist in the universe, it is estimated that researchers only know the chemical structures of 5% of them. Identifying the chemical structures of the remaining 95% has proven extremely challenging because many molecules exhibit low abundance, are contained in small volumes (e.g., <10 nL), do not readily ionize, exhibit similar structures to other molecules, etc. No single analytical technique exists to definitively identify the structure of an unknown molecule, and thus multiple different molecular measurements are typically made (i.e., multi-modal approach). Ion mobility (IMS) and mass spectrometry (MS) are two key tools that researchers use to determine the chemical structures of unknown molecules, and recently high-resolution and ultrahigh resolution IMS-MS instruments have provided greater confidence than ever before. However, HR-IMS-MS instruments typically exhibit low ion utilization efficiency, meaning they require large amounts of sample for an analysis (e.g., >10 µL). Unfortunately, this limitation prohibits the analysis of small volume samples where many unknown molecules exist. Described herein are the efforts made to enable the analysis of ultralow volumes with an HR-IMS-MS platform. A new scanning technique, termed a ‘stuttered traveling wave scan’, was developed as a replacement for the dual-gated scanning technique and works by halting the traveling waves after allowing ions to separate and then repeatedly restarting and stopping the traveling waves to incrementally move ions from the SLIM to the Orbitrap. Ions were stored inside the SLIM while the TWs were stopped, allowing the Orbitrap to perform high-resolution mass analysis. When the Orbitrap was ready, the TWs were restarted for short periods of time (<10 ms) to move ions from the SLIM to the Orbitrap. It was discovered that lower TW amplitudes and speeds than used during IMS separation were required to produce IMS peaks with the highest signal intensities and best resolving powers. The stuttered TW scan was found to produce similar resolutions and signal intensities compared to the dual-gated scanning technique. A new IMS design possessing an intersecting ‘tee’ with a reversible traveling wave was also developed to improve ion utilization efficiency during cyclic operation, which is necessary when only a single IMS spectrum can be acquired, such as when analyzing ultralow volume samples. The new capabilities described in this report lay the groundwork for acquiring HR-IMS-MS spectra of ultralow volume biological samples, such as single cells, where HR-IMS-MS can help elucidate the structures of unknown compounds.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Centriole and transition zone structures in photoreceptor cilia revealed by cryo-electron tomography

Primary cilia mediate sensory signaling in multiple organisms and cell types but have structures adapted for specific roles. Structural defects in them lead to devastating diseases known as ciliopathies in humans. Key to their functions are structures at their base: the basal body, the transition zone, the “Y-shaped links,” and the “ciliary necklace.” We have used cryo-electron tomography with subtomogram averaging and conventional transmission electron microscopy to elucidate the structures associated with the basal region of the “connecting cilia” of rod outer segments in mouse retina. The longitudinal variations in microtubule (MT) structures and the lumenal scaffold complexes connecting them have been determined, as well as membrane-associated transition zone structures: Y-shaped links connecting MT to the membrane, and ciliary beads connected to them that protrude from the cell surface and form a necklace-like structure. These results represent a clearer structural scaffold onto which molecules identified by genetics, proteomics, and superresolution fluorescence can be placed in our emerging model of photoreceptor sensory cilia.

59 BASIC BIOLOGICAL SCIENCES↗

Shot noise sets the limit of quantification in electrochemical measurements

Detection of single molecules, particles, and rapid redox events is a challenge of electrochemical investigations and requires either an amplification strategy or significant averaging for the electrochemical current to exceed the noise level. We consider the minimum number of electrons required to reach the limit of quantification in these electrochemical measurements. A survey of the literature indicates that the state-of-the-art limit in current detection for different types of measurements (e.g. voltammetry, single-molecule redox cycling, ion channel recordings of single molecules, metal nanoparticle collision, and phase nucleation) is independent of the nature of the measurement and increases linearly with reciprocal response time, Δt -1 , over ~5 orders of magnitude (from ~10 to ~10 6 s -1 ). This paper demonstrates that the practical limit of quantification requires cumulative measurement of ~2100 electrons during Δt and is determined by statistics of counting electrons, that is, the shot noise in the current.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Novel small molecule FGF 23 inhibitors increase serum phosphate and improve skeletal abnormalities in Hyp mice

We report that excess fibroblast growth factor 23 (FGF23) causes hereditary hypophosphatemic rickets, such as X-linked hypophosphatemia (XLH) and tumor induced osteomalacia (TIO). A small molecule that specifically binds to FGF23 to prevent activation of the FGFR/a-Klotho complex has potential advantages over the currently approved systemically administered FGF23 blocking antibody. Using structure-based drug design we previously identified ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine) as a small molecule antagonist for FGF23. Additional structure-activity studies developed a series of ZINC13407541 analogues with enhanced drug-like properties. In this study, we tested in a pre-clinical Hyp mouse homologue of XLH a direct connect analogue (8n) [(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] that exhibited the greatest stability in microsomal assays, and 13a [(E)-2-((E)-4-methylstyryl)benzaldehyde oxime] that exhibited increased in vitro potency. Using cryo-electron microscopy (Cryo-EM) structure and computational docking, we identified a key binding residue (Q156) of the FGF23 antagonists, ZINC13407541 and its analogues (8n and 13a) in the N-terminal domain of FGF23 protein. Site-directed mutagenesis and bimolecular fluorescence complementation (BiFC)-fluorescence resonance energy transfer (FRET) assay confirmed the binding site of these three antagonists. We found that pharmacological inhibition of FGF23 with either of these compounds blocked FGF23 signaling and increased serum phosphate and 1,25(OH)2D concentrations in Hyp mice. Long-term parenteral treatment with 8n or 13a also enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate in Hyp mice. The more potent 13a compound had greater therapeutic effects in Hyp mice. Further optimization of these FGF23 inhibitors may lead to versatile drugs to treat excess FGF23-mediated disorders.

60 APPLIED LIFE SCIENCES↗

The Uranium-Containing and Thorium-Containing Anions Studied by Photoelectron Spectroscopy

An in-depth knowledge of actinide chemistry is fundamental to many aspects of nuclear science and technology, including the synthesis and processing of materials and the remediation of waste disposal sites. Among the actinides, the chemical bonding behaviors of actinium and thorium resemble those of the transition metals; the 5f-electrons of protactinium, uranium, neptunium, and plutonium often play important roles in their bonding; and among the still heavier elements, their bonding tends to mimic the lanthanide elements in terms of electron shielding and their f-electron contributions. Bonding that involves 5f-electrons, however, is especially important, in part because of the significance of uranium and plutonium, but also because these elements are among the few where f-electron participation in bonding is relatively common. This work focused on studying uranium-containing and thorium-containing anions in the gas phase using negative ion photoelectron spectroscopy. Since this technique directly probed valence electrons, it was uniquely positioned to address open questions regarding molecular bonding and electron configurations. A particularly important issue concerned how bonding in actinide-containing molecules was affected by modifications to their actinide atoms’ environment, i.e., due to their interaction with ligands. A closely related question was how actinide atoms’ suborbitals were qualitatively reordered and their energies quantitatively shifted as a result of their ligated environments. These were especially relevant issues in regard to uranium due to it having multiple possible oxidation states (OS) and the potential for 5f electron participation in bonding. The effects of ligands on oxidation states and 5f-orbital energies in uranium bonding was expected to be pronounced. Both ligands and excess electrons were seen as probes of actinide atoms within actinide-containing molecules. Our strategy for advancing knowledge of chemical bonding in the actinide-containing species utilized the synergy between experiments and theory, where in some cases experimental results validated theory and where in others computational results assisted in interpreting experiments. Calculations on actinide systems are terrifically challenging due to large spin-orbit interactions, relativistic effects, and just the sheer number of electrons involved. Even in the simplest species, e.g., U and U2, the most sophisticated, modern calculations carried out by the most experienced theorists often only approximate experimentally-measured values, such as electron affinities. For theory to provide confident predictions that can be used to solve real problems it needed an iterative and ultimately corrective mechanism by which its methods can develop further. Experiments can be used to identify when theory has failed; whereupon the subsequent process of using the experiment-theory interplay can be used to find the cause of the failure. Upon fixing it in one case, different test species can be proposed and studied by the experiment-theory combination to determine whether the problem has been corrected. Thus, experiments not only measure the values of molecular properties, they also provide navigational 3 beacons that keep computations off the reefs in an otherwise dark sea with few reference points. Experimental measurements in the actinide field are not only important, they are in actuality essential to computational progress. While it was not always possible to compare the theoreticallydetermined quantity of interest directly with the same experimentally-measured observable, it was usually possible to compare consequential properties that are both calculable and measurable. In the work completed here electron affinities and electronic state spacings were often sensitive consequential parameters. Reasonable agreement between measured and computational values signaled that a calculation that was very likely to be on-track. We had established collaborative relationships with five computational groups, all of which have expertise in computational actinide chemistry. Their PI’s are L. Cheng, D. Dixon, L. Gagliardi, K. Peterson, and B. Vlaisavljevich. Our close interaction with our theory partners led to us suggesting systems to them and them to us. This reciprocal interaction between our experimental and their computational results was among the most important strengths of this work and was a thread woven throughout. Even though anion photoelectron spectroscopic studies are conducted on anions, much of the information that they provide, pertains to the electronic structure of the neutral counterparts of those anions; among these are electron affinities and electronically excited state spacings. Our experimental tools included several specialized ion sources for forming the anionic species of interest, a mass spectrometer for identifying and mass-selecting them, and an anion photoelectron spectrometer for determining their electron affinities (EA) and characterizing the electronic states of the selected anions’ neutral counterparts. Anion photoelectron spectroscopy is conducted by crossing a mass-selected beam of anions with a fixed-frequency laser beam and energy-analyzing the resultant photodetached electrons. The photodetachment process is governed by the energyconserving relationship: hν = EBE + EKE, where hν is the photon’s energy, EBE is the electron binding (photodetachment transition) energy, and EKE is the electron’s kinetic energy. In our apparatus mass-selection is accomplished via time-of-flight mass spectrometry (TOF-MS), electron energy analysis is achieved with either a magnetic bottle or by velocity mapped imaging. Photodetachment of electrons from anions is implemented via either Nd:YAG or excimer lasers. The photodetachment transition energy, i.e., the EBE, between the ground vibrational and electronic state of an anion and the ground vibrational and electronic state of that anion’s neutral counterpart is the adiabatic electron affinity (EA) of that neutral molecule. Likewise, photodetachment transitions between the ground vibrational and electronic state of an anion and the various electronically-excited states of that anion’s corresponding neutral map the electronic spectrum of that neutral species, i.e., the spectral spacings in the photoelectron spectrum are a mirror image of the neutral’s electronic spectrum. It was, of course, crucial to be able to form the anionic species of interest. There, we had a particularly broad field of anion sources from which to choose. These included several variants of pulsed laser vaporization (LV), laser photoemission, infrared desorption plus photoemission, pulsed arc discharge (PACIS), electrospray ionization (ESI), and Rydberg electron transfer (RET). Each of these anion sources were readily combined with, i.e., connected to, the anion photoelectron spectroscopic portion of our apparatus as described above. Among the sources that utilize lasers, visible light for LV sources as well as IR for desorption sources are provided by Nd:YAG lasers. Ultraviolet photons are provided by both Nd:YAG and excimer lasers, whereas the excitation wavelengths for RET experiments come from two Nd:YAG-pumped dye lasers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Structural basis of divergent substrate recognition and inhibition of human neurolysin

A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1–13) potently inhibits the Nln activity while dynorphin A (1–8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.

59 BASIC BIOLOGICAL SCIENCES↗