Using in Situ X‑ray Reflectivity to Study Protein Adsorption on Hydrophilic and Hydrophobic Surfaces: Benefits and Limitations

We have employed in situ X-ray reflectivity (IXRR) to study the adsorption of a variety of proteins (lysozyme, cytochrome c, myoglobin, hemoglobin, serum albumin, and immunoglobulin G) on model hydrophilic (silicon oxide) and hydrophobic surfaces (octadecyltrichlorosilane self-assembled monolayers), evaluating this recently developed technique for its applicability in the area of biomolecular studies. We report herein the highest resolution depiction of adsorbed protein films, greatly improving on the precision of previous neutron reflectivity (NR) results and previous IXRR studies. We were able to perform complete scans in 5 min or less with the maximum momentum transfer of at least 0.52 Å^–1, allowing for some time-resolved information about the evolution of the protein film structure. The three smallest proteins (lysozyme, cytochrome c, and myoglobin) were seen to deposit as fully hydrated, nondenatured molecules onto hydrophilic surfaces, with indications of particular preferential orientations. Time evolution was observed for both lysozyme and myoglobin films. The larger proteins were not observed to deposit on the hydrophilic substrates, perhaps because of contrast limitations. On hydrophobic surfaces, all proteins were seen to denature extensively in a qualitatively similar way but with a rough trend that the larger proteins resulted in lower coverage. We have generated high-resolution electron density profiles of these denatured films, including capturing the growth of a lysozyme film. Because the solution interface of these denatured films is diffuse, IXRR cannot unambiguously determine the film extent and coverage, a drawback compared to NR. X-ray radiation damage was systematically evaluated, including the controlled exposure of protein films to high-intensity X-rays and exposure of the hydrophobic surface to X-rays before adsorption. Our analysis showed that standard measuring procedures used for XRR studies may lead to altered protein films; therefore, we used modified procedures to limit the influence of X-ray damage

Effects of Divalent Cations on Phase Behavior and Structure of a Zwitterionic Phospholipid (DMPC) Monolayer at the Air−Water Interface

Effects of divalent cations (Ca²⁺, Mg²⁺, Ni²⁺, and Zn²⁺) on a zwitterionic phospholipid monolayer at the air−water interface are investigated by surface pressure−area isotherms and in situ X-ray scattering. Divalent cations lower the surface pressure for the fluid (LE) to condensed (L₂) phase transition in a strongly ion-specific manner. Surprisingly, the two-dimensional lattice dimensions and the tilt of the lipids’ alkyl tails in the L₂ phase show a nearly ion-nonspecific dependence on the excess surface pressure above the transition pressure. An empirical “universal” relationship was found between the tail tilt and the excess pressure, with the tails in the L₂ phase always displaying a tilt of 29° at the transition. A practical implication of these results is that, regardless of the divalent cation present, the microscopic details of the lipid tail packing in the L₂ phase can be deduced at any surface pressure once the transition pressure is obtained from isotherms

Structure of Biodegradable Films at Aqueous Surfaces: X‑ray Diffraction and Spectroscopy Studies of Polylactides and Tyrosine-Derived Polycarbonates

Three representative polymers of increasing modulus, poly­(d,l-lactic acid), PDLLA, poly­(desaminotyrosyl-tyrosine ethyl ester carbonate), PDTEC, and the same polymer with iodinated DTE segments, PI₂DTEC, were characterized by surface-pressure versus area (Π–<i>A</i>) isotherms and surface sensitive X-ray diffraction techniques. Films of 10–100 Å thickness were prepared for these studies by spreading dilute polymer solutions at air–water interfaces. The general properties of the isotherms and the Flory exponents, determined from the isotherms, vary in accordance with the increasing modulus of PDLLA, PDTEC, PI₂DTEC, respectively. The analysis of in situ X-ray reflectivity and grazing incidence X-ray diffraction (GIXD) measurements from films at aqueous surfaces provides a morphological picture that is consistent with the modulus of the polymers, and to a large extent, with their packing in their dry-bulk state. Large absorption of X-rays by iodine enabled X-ray spectroscopic studies under near-total-reflection conditions to determine the iodine distribution in the PI₂DTEC film and complement the structural model derived from reflectivity and GIXD. These structural studies lay the foundation for future studies of polymer–protein interactions at aqueous interfaces

Monomolecular Siloxane Film as a Model of Single Site Catalysts

Achieving structurally well-defined catalytic species requires a fundamental understanding of surface chemistry. Detailed structural characterization of the catalyst binding sites <i>in situ</i>, such as single site catalysts on silica supports, is technically challenging or even unattainable. Octadecyl­trioxysilane (OTOS) monolayers formed from octadecyl­trimethoxysilane (OTMS) at the air–liquid interface after hydrolysis and condensation at low pH were chosen as a model system of surface binding sites in silica-supported Zn²⁺ catalysts. We characterize the system by grazing incidence X-ray diffraction, X-ray reflectivity (XR), and X-ray fluorescence spectroscopy (XFS). Previous X-ray and infrared surface studies of OTMS/OTOS films at the air–liquid interface proposed the formation of polymer OTOS structures. According to our analysis, polymer formation is inconsistent with the X-ray observations and structural properties of siloxanes; it is energetically unfavorable and thus highly unlikely. We suggest an alternative mechanism of hydrolysis/condensation in OTMS leading to the formation of structurally allowed cyclic trimers with the six-membered siloxane rings, which explain well both the X-ray and infrared results. XR and XFS consistently demonstrate that tetrahedral [Zn­(NH₃)₄]²⁺ ions bind to hydroxyl groups of the film at a stoichiometric ratio of OTOS:Zn ∼ 2:1. The high binding affinity of zinc ions to OTOS trimers suggests that the six-membered siloxane rings are binding locations for single site Zn/SiO₂ catalysts. Our results show that OTOS monolayers may serve as a platform for studying silica surface chemistry or hydroxyl-mediated reactions

X‑ray Reflectivity Reveals a Nonmonotonic Ion-Density Profile Perpendicular to the Surface of ErCl₃ Aqueous Solutions

Complex interactions that determine ionic ordering in the bulk of electrolyte solutions are modified by surface-region inhomogeneities. We present results from an investigation of surface-ionic profiles that provide insights into the underlying physical chemistry in this region. X-ray reflectivity measurements from the liquid surfaces of aqueous ErCl₃ solutions reveal in unprecedented detail a nonmonotonic electron density profile, which is interpreted in terms of a nonmonotonic surface distribution of cations (Er³⁺) and their relationship to the bulk. The combination of a heavy, multivalent Er³⁺ and a lighter, monovalent anion (Cl^–) results in a significant cation depletion layer at the surface followed by a subsurface region of notably enhanced Er³⁺. Studying a series of solutions as a function of solute concentration reveals marked changes in Er³⁺ distribution, the most notable of which are the depletion layer thickness variation from 7.8 Å at 0.2 M to 5.5 Å at 1.0 M and the damped, oscillatory, cation concentrations indicative of solute multilayering in the subsurface region. This nonmonotonic profile is consistent with an analysis of surface tension measurements by the Gibbs adsorption equation that predicts negative adsorption. Molecular dynamics simulations provide physical insight into the observed behavior, implicating the high charge on erbium for its nonmonotonic variation with depth. This work suggests that future studies employing higher-valent cations will enhance the understanding of liquid/vapor interfaces and their widespread importance in areas ranging from atmospheric chemistry to metal-ion separations

Some methods of solving mixed discrete analogues of elliptical boundary-value problems

The study deals with systems of linear algebraic equations of a special kind developing in mixed methods of finite elements. The work is aimed at studying into effective direct and iteration methods of solving the systems describing the problems of the theory of plates and shells. Conditions are received which are to be meeted by the numeration of the unknowns in the system to solve it with the use of the generalized Kholessky algorithm. Different kinds of predeterminants for the systems being studied are proposed; estimations of the spectrum of matrices are received. A program complex is developed for solving a system of a special kind by all the methods considered. The results are expedient to be used by specialists and scientific teams dealing with problems of the theory of plates and shellsAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Counterions under a Surface-Adsorbed Cationic Surfactant Monolayer: Structure and Thermodynamics

The surface adsorption of ionic surfactants is fundamental for many widespread phenomena in life sciences and for a wide range of technological applications. However, direct atomic-resolution structural experimental studies of noncrystalline surface-adsorbed films are scarce. Thus, even the most central physical aspects of these films, such as their charge density, remain uncertain. Consequently, theoretical models based on contradicting assumptions as for the surface films’ ionization are widely used for the description and prediction of surface thermodynamics. We employ X-ray reflectivity to obtain the Ångström-scale surface-normal structure of surface-adsorbed films of the cationic surfactant cetyltri­methyl­ammonium bromide (CTAB) in aqueous solutions at several different temperatures and concentrations. In conjunction with published neutron reflectivity data, we determine the surface-normal charge distribution due to the dissociated surfactants’ headgroups. The distribution appears to be inconsistent with the Gouy–Chapman model yet consistent with a compact Stern layer model of condensed counterions. The experimental surfactant adsorption thermodynamics conforms well to classical, Langmuir and Kralchevsky, adsorption models. Furthermore, the Kralchevsky model correctly reproduces the observed condensation of counterions, allowing the values of the adsorption parameters to be resolved, based on the combination of the present data and the published surface tension measurements

X-ray fluorescence from a model liquid/liquid solvent extraction system

X-ray fluorescence near total reflection (XFNTR) is measured from the liquid/liquid interface between dodecane and an ErCl3 aqueous solution by monitoring L shell Er emission lines. A custom sample cell is used to minimize absorption of the fluorescence x-rays that pass through dodecane on their way to the detector. The Er3+ concentration near the interface is related to the fluorescence intensity by a scale factor that is extracted by fitting the incident-angle dependent Er La emission line intensities for different ErCl3 bulk concentrations. As an application, we present the use of XFNTR to monitor the growth of interfacial crud in a model solvent extraction system consisting of an interface between a dodecane solution of bis(2-ethylhexyl) phosphate (HDEHP) and an ErCl3 aqueous solution. VC 2011 American Institute of Physics. [doi:10.1063/1.3661983

Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg²⁺ and Ca²⁺) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca²⁺ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration

Cyclization Improves Membrane Permeation by Antimicrobial Peptoids

The peptidomimetic approach has emerged as a powerful tool for overcoming the inherent limitations of natural antimicrobial peptides, where the therapeutic potential can be improved by increasing the selectivity and bioavailability. Restraining the conformational flexibility of a molecule may reduce the entropy loss upon its binding to the membrane. Experimental findings demonstrate that the cyclization of linear antimicrobial peptoids increases their bactericidal activity against <i>Staphylococcus aureus</i> while maintaining high hemolytic concentrations. Surface X-ray scattering shows that macrocyclic peptoids intercalate into Langmuir monolayers of anionic lipids with greater efficacy than for their linear analogues. It is suggested that cyclization may increase peptoid activity by allowing the macrocycle to better penetrate the bacterial cell membrane