Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Observation of Ion Electrosorption in Metal–Organic Framework Micropores with In Operando Small‐Angle Neutron Scattering

A molecular‐level understanding of transport and adsorption mechanisms of electrolyte ions in nanoporous electrodes under applied potentials is essential to control the performance of double‐layer capacitors. Here, in operando small‐angle neutron scattering (SANS) is used to directly detect ion movements into the nanopores of a conductive metal–organic framework (MOF) electrode under operating conditions. Neutron‐scattering data reveals that most of the void space within the MOF is accessible to the solvent. Upon the addition of the electrolyte sodium triflate (NaOTf), the ions are adsorbed on the outer surface of the protrusions to form a 30 Å layer instead of entering the ionophobic pores in the absence of an applied charging potential. The changes in scattering intensity when potentials are applied suggests the ion rearrangement in the micropores following different mechanisms depending on the electrode polarization. These observations shed insights on ion electrosorption in electrode materials.     

In Situ Small‐Angle X‐ray Scattering Investigation of the Formation of Dual‐Mesoporous Materials

The formation of a 2D‐hexagonal (p6m) silica‐based hybrid dual‐mesoporous material is investigated in situ by using synchrotron time‐resolved small‐angle X‐ray scattering (SAXS). The material is synthesized from a mixed micellar solution of a nonionic fluorinated surfactant, R^F₈(EO)₉ (EO=ethylene oxide) and a nonionic triblock copolymer, P123. Both mesoporous networks, with pore dimensions of 3.3 and 8.5 nm respectively, are observed by nitrogen sorption, transmission electron microscopy (TEM), and SAXS. The in situ SAXS experiments reveal that mesophase formation occurs in two steps. First the nucleation and growth of a primary 2D‐hexagonal network (N1), associated with mixed micelles containing P123, then subsequent formation of a second network (N2), associated with micelles of pure R^F₈(EO)₉. The data obtained from SAXS and TEM suggest that the N1 network is used as a nucleation center for the formation of the N2 network, which would result in the formation of a grain with two mesopore sizes. Understanding the mechanism of the formation of such materials is an important step towards the synthesis of more‐complex materials by fine tuning the porosity.     

Small‐Angle Neutron Scattering from Elastomeric Networks in which the Junctions Alternate Regularly in their Functionality

We compute scattering form factors for SANS from labeled paths in Gaussian phantom networks in which junctions alternate regularly in their functionality (the number of chains emanating from a junction). Our calculations are based on the James‐Guth model of rubber‐like elasticity, which assumes that fluctuations are strain independent, while mean vectors transform affinely with the applied strain. Kratky plots for scattering from isotropic and uniaxially stretched bifunctional networks are computed and compared with corresponding plots for the simpler unifunctional networks. The results show the effects of the length of the labeled path, extent of deformation, direction of scattering with respect to the principal axis of the deformation and the functionalities of the network junctions.

     

Small‐Angle Neutron Scattering Studies of Hemoglobin Confined Inside Silica Tubes of Varying Sizes

In addition to the chemical nature of the surface, the dimensions of the confining host exert a significant influence on confined protein structures; this results in immense biological implications, especially those concerning the enzymatic activities of the protein. This study probes the structure of hemoglobin (Hb), a model protein, confined inside silica tubes with pore diameters that vary by one order of magnitude (≈20–200 nm). The effect of confinement on the protein structure is probed by comparison with the structure of the protein in solution. Small‐angle neutron scattering (SANS), which provides information on protein tertiary and quaternary structures, is employed to study the influence of the tube pore diameter on the structure and configuration of the confined protein in detail. Confinement significantly influences the structural stability of Hb and the structure depends on the Si‐tube pore diameter. The high radius of gyration (R_g) and polydispersity of Hb in the 20 nm diameter Si‐tube indicates that Hb undergoes a significant amount of aggregation. However, for Si‐tube diameters greater or equal to 100 nm, the R_g of Hb is found to be in very close proximity to that obtained from the protein data bank (PDB) reported structure (R_g of native Hb=23.8 Å). This strongly indicates that the protein has a preference for the more native‐like non‐aggregated state if confined inside tubes of diameter greater or equal to 100 nm. Further insight into the Hb structure is obtained from the distance distribution function, p(r), and ab initio models calculated from the SANS patterns. These also suggest that the Si‐tube size is a key parameter for protein stability and structure.     

Light,Small Angle Neutron and X-Ray Scattering from Gels

This paper gives two examples of experiments that demonstrate the power of small angle scattering techniques in the study of swollen polymer networks. First, it is shown how the partly ergodic character of these systems is directly detected by neutron spin echo experiments. The observed total field correlation function of the intensity scattered from a neutral gel allows the ergodic contribution to be directly distinguished from the non ergodic part, at values of transfer wave vector q that lie well beyond the range of dynamic light scattering. The results can be compared with those obtained at much lower q from visible light scattering. Second, a recent application of small angle X-ray (SAXS) and neutron (SANS) scattering is described for a polyelectrolyte molecule, DNA, in semi-dilute solutions under near-physiological conditions. For these observations, the divalent ion normally present, calcium, is replaced by an equivalent ion, strontium. The comparison between SANS and SAXS yields a quantitative picture of the cloud of divalent counter-ions around the central DNA core. At physiological conditions, the cloud is thinner than that predicted on the basis of the Debye screening length but thicker than if the counter-ions were condensed on the DNA chain.     

Catenation and Aggregation of Multi‐Cavity Coordination Cages

A series of metal‐mediated cages, having multiple cavities, was synthesized from Pd^II cations and tris‐ or tetrakis‐monodentate bridging ligands and characterized by NMR spectroscopy, mass spectrometry, and X‐ray methods. The peanut‐shaped [Pd₃L¹₄] cage deriving from the tris‐monodentate ligand L¹ could be quantitatively converted into its interpenetrated [5Cl@Pd₆L¹₈] dimer featuring a linear {[Pd‐Cl‐]₅Pd} stack as an unprecedented structural motif upon addition of chloride anions. Small‐angle neutron scattering (SANS) experiments showed that the cigar‐shaped assembly with a length of 3.7 nm aggregates into mono‐layered discs of 14 nm diameter via solvophobic interactions between the hexyl sidechains. The hepta‐cationic [5Cl@Pd₆L¹₈] cage was found to interact with polyanionic oligonucleotide double‐strands under dissolution of the aggregates in water, rendering the compound class interesting for applications based on non‐covalent DNA binding.     

Structural Characterization of Biomaterials by Means of Small Angle X-rays and Neutron Scattering (SAXS and SANS), and Light Scattering Experiments

Scattering techniques represent non-invasive experimental approaches and powerful tools for the investigation of structure and conformation of biomaterial systems in a wide range of distances, ranging from the nanometric to micrometric scale. More specifically, small-angle X-rays and neutron scattering and light scattering techniques represent well-established experimental techniques for the investigation of the structural properties of biomaterials and, through the use of suitable models, they allow to study and mimic various biological systems under physiologically relevant conditions. They provide the ensemble averaged (and then statistically relevant) information under in situ and operando conditions, and represent useful tools complementary to the various traditional imaging techniques that, on the contrary, reveal more local structural information. Together with the classical structure characterization approaches, we introduce the basic concepts that make it possible to examine inter-particles interactions, and to study the growth processes and conformational changes in nanostructures, which have become increasingly relevant for an accurate understanding and prediction of various mechanisms in the fields of biotechnology and nanotechnology. The upgrade of the various scattering techniques, such as the contrast variation or time resolved experiments, offers unique opportunities to study the nano- and mesoscopic structure and their evolution with time in a way not accessible by other techniques. For this reason, highly performant instruments are installed at most of the facility research centers worldwide. These new insights allow to largely ameliorate the control of (chemico-physical and biologic) processes of complex (bio-)materials at the molecular length scales, and open a full potential for the development and engineering of a variety of nano-scale biomaterials for advanced applications.     

Comparative Study of Scattering and Osmotic Properties of Synthetic and Biopolymer Gels

Summary: The effect of monovalent/divalent cation exchange on the structure and osmotic properties of chemically cross-linked polyacrylate and DNA gels swollen in near physiological salt solutions has been investigated. Both systems exhibit a reversible volume phase transition in the presence of calcium ions. The small-angle neutron scattering spectra of these gels display qualitatively similar features. At low values of q surface scattering is observed, while in the intermediate q range the signal is characteristic of scattering from rod-like elements. At high values of q the scattering intensity is governed by the local (short-range) geometry of the polymer chains. The competition between monovalent and divalent cations has been studied by anomalous small-angle X-ray scattering (ASAXS). The ASAXS results reveal that the local concentration of the divalent counter-ions in the vicinity of the polymer chains significantly exceeds that of the monovalent counter-ions.     

Raman and Neutron Scattering Study of Partially Deuterated L‐Alanine: Evidence of a Solid‐Solid Phase Transition

Raman and neutron experiments using specific isotope labeling were combined in order to study the dynamics and structure of L ‐alanine. Inelastic neutron and Raman scattering data of C₂H₄(ND₂)CO₂D are discussed in relation to the doubling of the lattice parameter a observed by means of neutron powder diffraction in C₂D₄ (NH₂)CO₂H. The major changes accompanying the phase transition are found in the vibrational frequencies involving the torsional vibration τ(CO₂^?), which is clearly affected by the hydrogen bonds between the protons of the ammonium group and the oxygen atoms of the carboxylate group. At lower temperatures the rearrangement of identifiable hydrogen bonds induces changes in the bending vibration δ(ND₃), confirming some orientational disorder.     

Counterion Association and Structural Conformation Change of Charged PAMAM Dendrimer in Aqueous Solutions Revealed by Small Angle Neutron Scattering

Our previous study of the structure change of poly(amidoamine) starburst dendrimers (PAMAM) dendrimer of generation 5 (G5) have demonstrated that although the overall molecular size is practically unaffected by increasing DCl concentration, a configurational transformation, from a diffusive density profile to a more uniform density distribution, is clearly observed. In the current paper, the focus is placed on understanding the effect of counterion identity on the inter-molecular structure and the conformational properties by studying the effect due to DBr using small angle neutron scattering (SANS) and integral equation theory. While the overall molecular size is found to be essentially unaffected by the change in the pD of solutions, it is surprising that the intra-molecular configurational transformation is not observed when DBr is used. The overall effective charge of a dendrimer is nearly the same for α < 1, independent of the type of acids. However, when α > 1, the effect of counterion identity becomes significant, the effective charge carried by a charged G5 PAPAM protonated by DBr becomes smaller than that of solutions with DCl. As a consequence, a counterion identity dependence of counterion association is revealed: Under the same level of molecular protonation, the specific counterion association, which is defined as the ratio of bound chloride anions to positively charged amines per molecule, is larger for the G5 PAMAM dendrimer charged by DBr than the one by DCl.     

Small‐Angle X‐Ray Scattering and Near‐Infrared Vibrational Spectroscopy of Water Confined in Aerosol‐OT Reverse Micelles

The state of water confined in Aerosol‐OT–hydrocarbon–water reverse micelles with cyclohexane, n‐pentane, n‐octane, and n‐dodecane as apolar solvents is investigated by small‐angle X‐ray scattering and near‐infrared vibrational spectroscopy of the first overtone of the OH stretching mode of water. The experiments focus on water/AOT molecular ratios W₀=2–20, where water is strongly affected by the confinement and surface–water interactions. The pair‐distance distribution functions derived from the small‐angle scattering patterns allows a detailed characterization of the topology of these systems, and they indicate deviations from monodisperse, spherical water pools for some of these hydrocarbon systems. In contrast to a common assumption, the pool size does not scale linearly with W₀ in going from dry reverse micelles (W₀→0) to essentially bulk‐like water (W₀>20). The first overtone of the OH‐stretching vibration exhibits highly structured spectra, which reveal significant changes in the hydrogen bonding environment upon confinement. The spectra are rationalized by a core/shell model developed by Fayer and co‐workers. This model subdivides water into core water in the interior of the micelle and shell water close to the interface. Core water is modelled by the properties of bulk water, while the properties of shell water are taken to be those of water at W₀=2. The model allows the representation of the spectra at any hydration level as a linear combination of the spectra of core and shell water. Different approaches are critically reviewed and discussed as well.     

Ammonothermal Synthesis,X-Ray and Time-of-Flight Neutron Crystal-Structure Determination,and Vibrational Properties of Barium Guanidinate,Ba(CN3H4)2

We report the crystal structure of Ba(CN₃H₄)₂ as synthesized from liquid ammonia. Structure solution based on X-ray diffraction data suffers from a severe pseudo-tetragonal problem due to extreme scattering contrast, so the true monoclinic symmetry is detectable only from neutron powder diffraction patterns, and structure solution and refinement was greatly aided by density-functional theory. The symmetry lowering is due to slight deviations of the guanidinate anion from the mirror plane in space group P b2, a necessity of hydrogen bonding. At 300 K, barium guanidinate crystallizes in P2₁/c with a=6.26439(2) Å, b=16.58527(5) Å, c=6.25960(2) Å, and a monoclinic angle of β=90.000(1)°. To improve the data-to-parameter ratio, anisotropic displacement parameters from first-principles theory were incorporated in the neutron refinement. Given the correct structural model, the positional parameters of the heavy atoms were also refinable from X-ray diffraction of a twinned crystal. The two independent guanidinate anions adopt the all-trans- and the anti-shape. The Ba cation is coordinated by eight imino nitrogens in a square antiprism with Ba−N contacts between 2.81 and 3.04 Å. The IR and Raman spectra of barium guanidinate were compared with DFT-calculated phonon spectra to identify the vibrational modes.