Solution-phase Structures Research Articles

PW-SMD: A Plane-Wave Implicit Solvation Model Based on Electron Density for Surface Chemistry and Crystalline Systems in Aqueous Solution.

Electron density-based implicit solvation models are a class of techniques for quantifying solvation effects and calculating free energies of solvation without an explicit representation of solvent molecules. Integral to the accuracy of solvation modeling is the proper definition of the solvation shell separating the solute molecule from the solvent environment, allowing for a physical partitioning of the free energies of solvation. Unlike state-of-the-art implicit solvation models for molecular quantum chemistry calculations, e.g., the solvation model based on solute electron density (SMD), solvation models for systems under periodic boundary conditions with plane-wave (PW) basis sets have been limited in their accuracy. Furthermore, a unified implicit solvation model with both homogeneous solution-phase and heterogeneous interfacial structures treated on equal footing is needed. In order to address this challenge, we developed a high-accuracy solvation model for periodic PW calculations that is applicable to molecular, ionic, interfacial, and bulk-phase chemistry. Our model, PW-SMD, is an extension of the SMD molecular solvation model to periodic systems in water. The free energy of solvation is partitioned into the electrostatic and cavity-dispersion-solvent structure (CDS) contributions. The electrostatic contributions of the solvation shell surrounding solute structures are parametrized based on their geometric and physical properties. In addition, the nonelectrostatic contribution to the solvation energy is accounted for by extending the CDS formalism of SMD to incorporate periodic boundary conditions. We validate the accuracy and robustness of our solvation model by comparing predicted solvation free energies against experimental data for molecular and ionic systems, carved-cluster composite energetic models of solvated reaction energies and barriers on surface systems, and deep-learning-accelerated ab initio molecular dynamics (AIMD). Our developed periodic implicit solvation model shows significantly improved accuracy compared to previous work (namely, solvation models in aqueous solution) and can be applied to simulate solvent effects in a wide range of surface and crystalline materials.

Understanding the Vibrational Structure and Ultrafast Dynamics of the Metal Carbonyl Precatalyst [Mn(ppy)(CO)4

The solution phase structure, vibrational spectroscopy, and ultrafast relaxation dynamics of the precatalyst species [Mn(ppy)(CO)4] (1) in solution have been investigated using ultrafast two-dimensional infrared (2D-IR) spectroscopy. By comparing 2D-IR data with the results of anharmonic density functional theory (DFT) calculations, we establish an excellent agreement between measured and predicted inter-mode couplings of the carbonyl stretching vibrational modes of 1 that relates to the atomic displacements of axial and equatorial ligands in the modes and the nature of the molecular orbitals involved in M-CO bonding. Measurements of IR pump-probe spectra and 2D-IR spectra as a function of waiting time reveal the presence of ultrafast (few ps) intramolecular vibrational energy redistribution between carbonyl stretching modes prior to vibrational relaxation. The vibrational relaxation times of the CO-stretching modes of 1 are found to be relatively solvent-insensitive, suggestive of limited solvent-solute interactions in the ground electronic state. Overall, these data provide a detailed picture of the complex potential energy surface, bonding and vibrational dynamics of 1, establishing a fundamental basis for the next steps in understanding and modulating precatalyst behavior.

Structural Elucidation of Ubiquitin via Gas-Phase Ion/Ion Cross-Linking Reactions Using Sodium-Cationized Reagents Coupled with Infrared Multiphoton Dissociation.

Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N-hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

Decoding the Reactivity Enhancement of Ln2Ce2O7 Compounds (Ln = Yb, Y, Tb, and Gd) for Soot Combustion: The Remarkable Contribution of Variable Valence A-Sites.

The impact of variable valence A-sites on the redox property and reactivity of Ln2Ce2O7 compounds in soot particulate combustion has been investigated. It was observed that Yb2Ce2O7, Y2Ce2O7, and Gd2Ce2O7 formed a rare earth C-type phase, while Tb2Ce2O7 formed a solid solution phase. Both Tb2Ce2O7 and Yb2Ce2O7 possess dual valence state A-sites, resulting in significantly more surface vacancies. Additionally, the advantageous solid solution phase structure of Tb2Ce2O7 leads to even more surface vacancies than Yb2Ce2O7, which is crucial to generate active oxygen sites. Moreover, the introduction of NO into the reaction feed enhances combustion activity by producing active surface monodentate nitrates. A catalyst with higher numbers of surface vacancies exhibits improved NO oxidation ability and better NO2 utilization efficiency. Consequently, the Tb2Ce2O7 compound demonstrates not only the best soot combustion activity, but also an optimal NOx-assistance effect. Therefore, it is concluded that variable valence A-site is the intrinsic factor to improve the reactivity of Ln2Ce2O7 catalysts.

Microstructural Evolution and Mechanical Properties of Y Added CoCrFeNi High-entropy Alloys Produced by Arc-melting

The CoCrFeNi high entropy alloy (HEA) with face-centered cubic (FCC) crystal structure exhibits excellent ductility values even at cryogenic temperatures. However, since this HEA is relatively weak in strength, it may not meet the requirements of industrial applications in terms of strength-ductility trade-off. Therefore, the systematic addition of yttrium (Y) into CoCrFeNi HEA was investigated in the present study to increase the strength by solid solution and second phase strengthening. The HEAs were produced by vacuum arc melting, suction casting, and subsequent homogenization at 1150 °C for 24 h. The structural development of the HEAs was investigated by using the X-ray diffraction (XRD) technique revealing the formation of a solid solution phase and Ni3Y-type hexagonal structure (HS) second phase. The corresponding microstructure of the HEAs was examined under a scanning electron microscope (SEM) revealing the transformation of the microstructure from elongated grains to nearly equiaxed grains with the increase of Y content from 2 at. % to 4 at. %. The mechanical properties of the HEAs were investigated by using hardness and compression tests. The results exhibited a dramatic increase in the hardness from 143 (±2) HV to 335 (±7) HV and in the yield strength from 130 MPa to 1025 MPa with 4 at. % Y addition. Our study has revealed that the addition of rare earth Y element results in further development in the strength of the CoCrFeNi for potential engineering applications.

The effect of host size on binding in host-guest complexes of cyclodextrins and polyoxometalates.

Harnessing flexible host cavities opens opportunities for the design of novel supramolecular architectures that accommodate nanosized guests. This research examines unprecedented gas-phase structures of Keggin-type polyoxometalate PW12O40 3- (WPOM) and cyclodextrins (X-CD, X = α, β, γ, δ, ε, ζ) including previously unexplored large, flexible CDs. Using ion mobility spectrometry coupled to mass spectrometry (IM-MS) in conjunction with molecular dynamics (MD) simulations, we provide first insights into the binding modes between WPOM and larger CD hosts as isolated structures. Notably, γ-CD forms two distinct structures with WPOM through binding to its primary and secondary faces. We also demonstrate that ε-CD forms a deep inclusion complex, which encapsulates WPOM within its annular inner cavity. In contrast, ζ-CD adopts a saddle-like conformation in its complex with WPOM, which resembles its free form in solution. More intriguingly, the gas-phase CD-WPOM structures are highly correlated with their counterparts in solution as characterized by nuclear magnetic resonance (NMR) spectroscopy. The strong correlation between the gas- and solution phase structures of CD-WPOM complexes highlight the power of gas-phase IM-MS for the structural characterization of supramolecular complexes with nanosized guests, which may be difficult to examine using conventional approaches.

Spontaneous and Selective Macrocyclization in Nitroaldol Reaction Systems.

Through a dynamic polymerization and self-sorting process, a range of lowellane macrocycles have been efficiently generated in nitroaldol systems composed of aromatic dialdehydes and aliphatic or aromatic dinitroalkanes. All identified macrocycles show a composition of two repeating units, resulting in tetra-β-nitroalcohols of different structures. The effects of the building block structure on the macrocyclization process have been demonstrated, and the influence from the solvent has been explored. In general, the formation of the lowellanes was amplified in response to phase-change effects, although solution-phase structures were, in some cases, favored.

Are the Gas-Phase Structures of Molecular Elephants Enduring or Ephemeral? Results from Time-Dependent, Tandem Ion Mobility.

The structural stability of biomolecules in the gas phase remains an important topic in mass spectrometry applications for structural biology. Here, we evaluate the kinetic stability of native-like protein ions using time-dependent, tandem ion mobility (IM). In these tandem IM experiments, ions of interest are mobility-selected after a first dimension of IM and trapped for up to ∼14 s. Time-dependent, collision cross section distributions are then determined from separations in a second dimension of IM. In these experiments, monomeric protein ions exhibited structural changes specific to both protein and charge state, whereas large protein complexes did not undergo resolvable structural changes on the timescales of these experiments. We also performed energy-dependent experiments, i.e., collision-induced unfolding, as a comparison for time-dependent experiments to understand the extent of unfolding. Collision cross section values observed in energy-dependent experiments using high collision energies were significantly larger than those observed in time-dependent experiments, indicating that the structures observed in time-dependent experiments remain kinetically trapped and retain some memory of their solution-phase structure. Although structural evolution should be considered for highly charged, monomeric protein ions, these experiments demonstrate that higher-mass protein ions can have remarkable kinetic stability in the gas phase.

Diserinol Isophthalamide: A Novel Reagent for Complexation with Biomolecular Anions in Electrospray Ionization Mass Spectrometry

Transferring biomolecules from solution to vacuum facilitates a detailed analysis of molecular structure and dynamics by isolating molecules of interest from a complex environment. However, inherent in the ion desolvation process is the loss of solvent hydrogen bonding partners, which are critical for the stability of a condensed-phase structure. Thus, transfer of ions to vacuum can favor structural rearrangement, especially near solvent-accessible charge sites, which tend to adopt intramolecular hydrogen bonding motifs in the absence of solvent. Complexation of monoalkylammonium moieties (e.g., lysine side chains) with crown ethers such as 18-crown-6 can disfavor structural rearrangement of protonated sites, but no equivalent ligand has been investigated for deprotonated groups. Herein we describe diserinol isophthalamide (DIP), a novel reagent for the gas-phase complexation of anionic moieties within biomolecules. Complexation is observed to the C-terminus or side chains of the small model peptides GD, GE, GG, DF-OMe, VYV, YGGFL, and EYMPME in electrospray ionization mass spectrometry (ESI-MS) studies. In addition, complexation is observed with the phosphate and carboxylate moieities of phosphoserine and phosphotyrosine. DIP performs favorably in comparison to an existing anion recognition reagent, 1,1'-(1,2-phenylene)bis(3-phenylurea), that exhibits moderate carboxylate binding in organic solvent. This improved performance in ESI-MS experiments is attributed to reduced steric constraints to complexation with carboxylate groups of larger molecules. Overall, diserinol isophthalamide is an effective complexation reagent that can be applied in future work to study retention of solution-phase structure, investigate intrinsic molecular properties, and examine solvation effects.

Online protein unfolding characterized by ion mobility electron capture dissociation mass spectrometry: cytochrome C from neutral and acidic solutions.

Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation (ECD) of proteins ionized from aqueous solutions, have been used for the study of solution-like structures of intact proteins. By mixing aqueous proteins with denaturants online before ESI, the amount of protein unfolding can be precisely controlled and rapidly analyzed, permitting the characterization of protein folding intermediates in protein folding pathways. Herein, we mixed various pH solutions online with aqueous cytochrome C for unfolding and characterizing its unfolding intermediates with ESI-MS charge state distribution measurements, IMS, and ECD. The presence of folding intermediates and unfolded cytochrome c structures were detected from changes in charge states, arrival time distributions (ATDs), and ECD. We also compared structures from nondenaturing and denaturing solution mixtures measured under "gentle" (i.e., low energy) ion transmission conditions with structures measured under "harsh" (i.e., higher energy) transmission. This work confirms that when using "gentle" instrument conditions, the gas-phase cytochrome c ions reflect attributes of the various solution-phase structures. However, "harsh" conditions that maximize ion transmission produce extended structures that no longer correlate with changes in solution structure.

Native Top-Down Mass Spectrometry with Collisionally Activated Dissociation Yields Higher-Order Structure Information for Protein Complexes.

Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry, but elucidating structures to understand their function is more challenging. Native top-down MS (nTDMS), i.e., fragmentation of the gas-phase protein, is conventionally used to derive sequence information, locate post-translational modifications (PTMs), and pinpoint ligand binding sites. nTDMS also endeavors to dissociate covalent bonds in a conformation-sensitive manner, such that information about higher-order structure can be inferred from the fragmentation pattern. However, the activation/dissociation method used can greatly affect the resulting information on protein higher-order structure. Methods such as electron capture/transfer dissociation (ECD and ETD, or ExD) and ultraviolet photodissociation (UVPD) can produce product ions that are sensitive to structural features of protein complexes. For multi-subunit complexes, a long-held belief is that collisionally activated dissociation (CAD) induces unfolding and release of a subunit, and thus is not useful for higher-order structure characterization. Here we show not only that sequence information can be obtained directly from CAD of native protein complexes but that the fragmentation pattern can deliver higher-order structural information about their gas- and solution-phase structures. Moreover, CAD-generated internal fragments (i.e., fragments containing neither N-/C-termini) reveal structural aspects of protein complexes.

Studies related to changes in ionic strength due to addition of sodium chloride to an aqueous solution of cationic dye methylene blue (MB): Electronic spectra analysis for dimerization equilibria

It is well-known that methylene blue (MB),a cationic dye forms dimer and higher aggregates in aqueous solutions. For specific concentration range (1∙10−6–1∙10−4 M), a D⇌2M equilibria has been postulated and well characterized by visible electronic spectroscopy. The dye molecules can lead to formation of stacking (end on end and sandwich) type structures in solution phase stabilized by weak dispersion van der Waals forces and cation-anion-cation ion pairs and triplet interactions usually referred as hydrophobic interactions. Recent literature reports indicate that excess chemical potential of small hydrophobic particles and the strength of hydrophobic pair interactions appear to be linearly related. Our observations about precipitation of dye (MB) at higher concentrations of salt like sodium chloride (NaCl) made us to study the dimerization equilibria in detail since in the Corona health disaster; use of MB to diagnose the type of Covid variants helped Scientists in a great way. Salting-out of proteins is in general used to explain the specific protein aggregation behaviour observed in Huntington's disease. For model compound like tertiary butyl-alcohol, Geiger et al. reported enhancement of hydrophobic contacts which increase with increase in salt concentration. NaCl is an essential ingredient of biological fluids, of which concentration controls the various biological life governing processes. However, such information is non-existent for aqueous solutions of dyes.We therefore reported measured optical absorbance of aqueous solution containing NaCl (0.1–1.0 M) and dye at different concentrations (1∙10−6–1∙10−4 M) at 298 K spectrophotometrically, in the wavelength range of 550–700 nm. The spectrums are compared to those obtained for binary solutions of MB in water. Analysis of the spectral data yielded the dimer dissociation constant and individual characteristic monomer and dimer spectra. Further information about the geometry and twist angle between the dipoles of the MB molecules in the dimer species has been obtained. The results are analyzed for the monomer-dimer equilibria and salting-out effect as well applications of mass action model and exciton theories are discussed.

Convenient synthesis of dipeptide structures in solution phase assisted by a thioaza functionalized magnetic nanocatalyst

In this study, a heterogeneous nanocatalyst is presented that is capable to efficiently catalyze the synthetic reactions of amide bond formation between the amino acids. This nanocatalyst which is named Fe3O4@SiO2/TABHA (TABHA stands for thio-aza-bicyclo-hepten amine), was composed of several layers that increased the surface area to be functionalized with 2-aminothiazole rings via Diels–Alder approach. Firstly, various analytic methods such as Fourier-transform infrared (FTIR) and energy-dispersive X-ray (EDX) spectroscopic methods, thermogravimetric analysis (TGA), electron microscopy (EM), and UV–vis diffuse reflectance spectroscopy (UV-DRS) have been used to characterize the desired structure of the Fe3O4@SiO2/TABHA catalyst. Afterward, the application of the presented catalytic system has been studied in the peptide bond formation reactions. Due to the existence of a magnetic core in the structure of the nanocatalyst, the nanoparticles (NPs) could be easily separated from the reaction medium by an external magnet. This special feature has been corroborated by the obtained results from vibrating-sample magnetometer (VSM) analysis that showed 24 emu g−1 magnetic saturation for the catalytic system. Amazingly, a small amount of Fe3O4@SiO2/TABHA particles (0.2 g) has resulted in ca. 90% efficiency in catalyzing the peptide bond formation at ambient temperature, over 4 h. Also, this nanocatalyst has demonstrated an acceptable recycling ability, where ca. 76% catalytic performance has been observed after four recycles. Due to high convenience in the preparation, application, and recyclization processes, and also because of lower cost than the traditional coupling reagents (like TBTU), the presented catalytic system is recommended for the industrial utilization.

Co-Construction of Solid Solution Phase and Void Space in Yolk-Shell Fe0.4Co0.6S@N-Doped Carbon to Enhance Cycling Capacity and Rate Capability for Aluminum-Ion Batteries.

Rechargeable aluminum-ion batteries (AIBs), using low-cost and inherent safety Al metal anodes, are regarded as promising energy storage devices next to lithium-ion batteries. Currently, one of the greatest challenges for AIBs is to explore cathodes suitable for feasible Al3+ insertion/extraction with high structure stability. Herein, a facile co-engineering on solid solution phase and cavity structure is developed via Prussian blue analogues by a simple and facile sulfidation strategy. The obtained uniform yolk-shell Fe0.4Co0.6S@N-doped carbon nanocages (y-s Fe0.4Co0.6S@NC) display a high reversible capacity of 141.3 mA h g-1 at 500 mA g-1 after 100 cycles and a good rate capability of 100.9 mA h g-1 at 1000 mA g-1. The improved performance can be mainly ascribed to the dual merits of the composite; that is, more negative Al3+ formation energy and improved Al3+ diffusion kinetics favored by the solid solution phase and Al3+ insertion/extraction accommodable space stemmed from the yolk-shell structure. Moreover, the reaction mechanism study discloses that the reaction involves the intercalation of Al3+ ions into Fe0.4Co0.6S to generate AllFemConS and elemental Fe and Co.

Improved thermal stability of AlCrSiN coatings base on the template effect of TiAlN layer

ABSTRACT TiAlN/AlCrSiN multilayer coatings were prepared by arc ion plating. The implements of various thickness ratio (0, 0.89, 1.04, 1.14 and 1.49) of TiAlN/AlCrSiN on microstructure, mechanical properties and thermal stability of TiAlN/AlCrSiN multilayer coatings were investigated. The AlCrSiN single coating without TiAlN inserted layer exhibits a mixed-phase structure of c-AlN and c-CrN, while TiAlN/AlCrSiN multilayer coatings show a single-phase cubic structure. As the thickness ratio of TiAlN/AlCrSiN increases, the critical load of multilayer coating gradually increases up to 99 N, the hardness decreases and the thermal stability improved. The TiAlN/AlCrSiN multilayer coating with a high thickness ratio of 1.49 was able to maintain a single (200) preferred c-TiAlN/AlCrN solid solution phase structure after annealing at 800°C in Ar for 2 h. The obtained single-phase cubic structure of AlCrSiN at room temperature and the improved thermal stability properties can be explained by the template effect of TiAlN on the AlCrSiN.

The complicating role of pnictogen bond formation in the solution-phase and solid-state structures of the heavier pnictogen atranes.

The structure of the simplest stibatrane has been a mystery since it was first prepared in 1966. This study reports the preparation and characterization of two stibatranes from triethanolamine and triisopropanolamine. Solid state structures reveal macrocycles that contain favourable inter- and intramolecular pnictogen bonds. Solution studies, corroborated by DFT analysis, reveal an equilibrium mixture assigned to monomer and pnictogen-bonded dimer. This allowed for the determination of an enthalpy associated with pnictogen bond formation of -27 kJ mol-1, in line with the supramolecular nature of these interactions.

Cover Picture: Bimetallic Copper/Ruthenium/Osmium Complexes: Observation of Conformational Differences Between the Solution Phase and Solid State by Atomic Pair Distribution Function Analysis (Angew. Chem. Int. Ed. 5/2022)

The solution-phase molecular structure of five redox- and photo-active bimetallic complexes was directly probed using high-energy X-ray scattering and atomic pair distribution function analysis in the Research Article by Xiaosong Li, Karen L. Mulfort et al. (e202111764). The analysis reveals significant conformational differences between the solution-phase and solid-state structures of these complexes that may be relevant for interactions between the two metal centers.

Impact of Ambient Vapors Present in an Electrospray Ionization Source on Gas-Phase Ion Structures.

According to current consensus, structures of protomeric (or deprotomeric) tautomers of gaseous ions generated by electrospray ionization depend primarily on the nature of the spray solvent. To probe the effect of the spray solvent on protonation, 4-aminobenzoic acid (PABA) has often been selected as the model compound. It is widely accepted that the protonation in the gas phase takes place primarily on the carbonyl oxygen atom when the sample is sprayed in methanol and on the nitrogen atom when acetonitrile is used as the spray solvent. Although this observation is valid, our current results indicate that the determination of the predominant protomer in the gas phase by the spray solvent is an indirect effect moderated by the solvent vapor molecules present in the ambient ion source. To investigate real-time changes in protomer distributions due to solvents, we used ion-mobility mass spectrometry (IM-MS). Initially, when a PABA solution in methanol was electrosprayed, the ion-mobility arrival-time profile recorded showed essentially one peak for the O-protomer. However, when acetonitrile or acetone vapors were introduced to the ambient-pressure ion source via the flowing desolvation gas, the intensity of the O-protomer peak diminished rapidly, and the N-protomer signal became dominant. The moment the acetonitrile (or acetone) vapors were removed from the ion source, the protomer-distribution signals began gradually reverting back to their original intensities. Furthermore, when PABA samples in methanol and acetonitrile were electrosprayed separately via a dual-sprayer setup, which allowed for the selective blocking of the gaseous ion-generation cascade of charged droplets from either sprayer, the predominant signal corresponded only to the N-protomer, irrespective of the position of the mechanical barrier. Because the mechanical barrier prevents only the gaseous ion formation, but not the physical access of solvent vapors to the ion source, it is evident that the solvent vapor that engulfs the ion source is the governing factor that decides the protomer distribution, not the nature of the spray solvent. Noticeably, acetonitrile wields a stronger effect on the manifested protomer distribution than many other solvents, including methanol, water, hexanes, and toluene. Apparently, the so-called "memory" of the solution-phase structures and the phenomenon described as "kinetic trapping" are both due to indirect effects caused by the solvent vapor engulfing the atmospheric-pressure ion source. Moreover, the so-called "memory" effect can either be "saved" or "erased" by exposing the initially formed gaseous ions to different solvent vapors from an alternative source.

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis-Trans Isomerization and the Chameleonic Roles of Calcium.

Falling outside of Lipinski's rule of five, macrocyclic drugs have accessed unique binding sites of their target receptors unreachable by traditional small molecules. Cyclosporin(e) A (CycA), an extensively studied macrocyclic natural product, is an immunosuppressant with undesirable side effects such as electrolytic imbalances. In this work, a comprehensive view on the conformational landscape of CycA, its interactions with Ca2+, and host-guest interactions with cyclophilin A (CypA) is reported through exhaustive analyses that combine ion-mobility spectrometry-mass spectrometry (IMS-MS), nuclear magnetic resonance (NMR) spectroscopy, distance-geometry modeling, and NMR-driven molecular dynamics. Our IMS-MS data show that CycA can adopt extremely compact conformations with significantly smaller collisional cross sections than the closed conformation observed in CDCl3. To adopt these conformations, the macrocyclic ring has to twist and bend via cis-trans isomerization of backbone amides, and thus, we termed this family of structures the "bent" conformation. Furthermore, NMR measurements indicate that the closed conformation exists at 19% in CD3OD/H2O and 55% in CD3CN. However, upon interacting with Ca2+, in addition to the bent and previously reported closed conformations of free CycA, the CycA:Ca2+ complex is open and has all-trans peptide bonds. Previous NMR studies using calcium perchlorate reported only the closed conformation of CycA (which contains one cis peptide bond). Here, calcium chloride, a more biologically relevant salt, was used, and interestingly, it helps converting the cis-MeLeu9-MeLeu10 peptide bond into a trans bond. Last, we were able to capture the native binding of CycA and CypA to give forth evidence that IMS-MS is able to probe the solution-phase structures of the complexes and that the Ca2+:CycA complex may play an essential role in the binding of CycA to CypA.

Solution Structure of the Gloeobacter Violaceus Ligand-Gated Ion Channel Probed by Small-Angle Neutron Scattering

Pentameric ligand-gated ion channels transform chemical signals into electrical ones, a process during which they undergo subtle conformational cycling resulting in rapid, reversible gating of an intrinsic transmembrane pore. The pH-gated bacterial channel GLIC has proved a valuable model system for this receptor family, in part due to its accessibility to X-ray crystallography - which has provided structures of closed, open, and intermediate conformations. However, such models raise critical questions as to whether the X-ray structures represent actual functional states, and if the cycle includes additional conformations such as highly contracted or expanded states. Small-angle neutron scattering (SANS) offers an approach to characterizing solution-phase structure, increasingly applicable to biomolecular problems on this scale. Here we characterized GLIC by SANS, in combination with inline size-exclusion chromatography (SEC), match-out deuterated detergents, and computational modeling of multiple putative structural models. SEC-SANS under resting conditions (pH 7) enabled the characterization of non-aggegated species which fit predictions from resting-state models, particularly following unrestrained molecular dynamics simulation of X-ray structures. Curves collected under activating conditions (pH 3) best fit a mixture of resting- and open-state models, whereas they diverged from expanded-pore models. in addition to supporting less-expanded models of the open state, this work demonstrates that SEC-SANS, in combination with molecular modeling and simulations, can distinguish solution-phase functional states of an ion channel. As neutron source brilliance increases and inline SEC-SANS set-ups become increasingly accessible, such methods will offer valuable tools for the elucidation of receptor conformational cycling and pharmaceutical development.