Curtis W. Meuse

Hybrid bilayer membranes in air and water: infrared spectroscopy and neutron reflectivity studies.

In this report we describe the fabrication and characterization of a phospholipid/alkanethiol hybrid bilayer membrane in air. The bilayer is formed by the interaction of phospholipid with the hydrophobic surface of a self-assembled alkanethiol monolayer on gold. We have characterized the resulting hybrid bilayer membrane in air using atomic force microscopy, spectroscopic ellipsometry, and reflection-absorption infrared spectroscopy. These analyses indicate that the phospholipid added is one monolayer thick, is continuous, and exhibits molecular order which is similar to that observed for phospholipid/phospholipid model membranes. The hybrid bilayer prepared in air has also been re-introduced to water and characterized using neutron reflectivity and impedance spectroscopy. Impedance data indicate that when moved from air to water, hybrid bilayers exhibit a dielectric constant and thickness that is essentially equivalent to hybrid bilayers prepared in situ by adding phospholipid vesicles to alkanethiol monolayers in water. Neutron scattering from these samples was collected out to a wave vector transfer of 0.25 A(-1), and provided a sensitivity to changes in total layer thickness on the order of 1-2 A. The data confirm that the acyl chain region of the phospholipid layer is consistent with that observed for phospholipid-phospholipid bilayers, but suggest greater hydration of the phospholipid headgroups of HBMs than has been reported in studies of lipid multilayers.

Raman and FTIR Spectroscopies of Fluorescein in Solutions

Raman and Fourier transform-infra red (FT-IR) spectroscopies of fluorescein in aqueous solutions have been investigated in the pH range from 9.1 to 5.4. At pH 9.1 fluorescein is in the dianion form. At pH 5.4, fluorescein is a mixture of monoanion (approximately 85%), dianion and neutral forms (together approximately 15%). The fluorescence quantum yield drops from 0.93 for the dianion form to 0.37 for the monoanion form. The Raman and FT-IR studies focused on the frequency range from 1000 to 1800 cm(-1) which contains the skeletal vibrational modes of the xanthene moiety of fluorescein. At pH 9.1, the spectroscopic feature of fluorescein dianion are consistent with a picture of an electron delocalized among the xanthene moiety and two identical oxygens attached to opposite ends of the xanthene moiety, forming a very symmetric structure. The characteristic of fluorescein dianion is the presence of the phenoxide-like stretch at 1310 cm(-1). At pH 5.4, fluorescein monoanion has lost the symmetric structure characteristic of the dianion. The spectra of the monoanion have distinctive contributions from the phenolic bend at 1184 cm(-1). The assignments of the vibrational bands shown in Raman and FT-IR spectra are given based on both literature and the ab initio calculations at the Hartree-Fock level with HF/6-31 + +G* basis set. Excellent correlation is found between the experimental and calculated spectra.

First-Principles Determination of Hybrid Bilayer Membrane Structure by Phase-Sensitive Neutron Reflectometry

The application of a new, phase-sensitive neutron reflectometry method to reveal the compositional depth profiles of biomimetic membranes is reported. Determination of the complex reflection amplitude allows the related scattering length density (SLD) profile to be obtained by a first-principles inversion without the need for fitting or adjustable parameters. The SLD profile so obtained is unique for most membranes and can therefore be directly compared with the SLD profile corresponding to the chemical compositional profile of the film, as predicted, for example, by a molecular dynamics simulation. Knowledge of the real part of the reflection amplitude, in addition to enabling the inversion, makes it possible to assign a spatial resolution to the profile for a given range of wavevector transfer over which the reflectivity data are collected. Furthermore, the imaginary part of the reflection amplitude can be used as a sensitive diagnostic tool for recognizing the existence of certain in-plane inhomogeneities in the sample. Measurements demonstrating the practical realization of this phase-sensitive technique were performed on a hybrid bilayer membrane (self-assembled monolayer of thiahexa (ethylene oxide) alkane on gold and a phospholipid layer) in intimate contact with an aqueous reservoir. Analysis of the experimental results shows that accurate compositional depth profiles can now be obtained with a spatial resolution in the subnanometer range, primarily limited by the background originating from the reservoir and the roughness of the films supporting substrate.

Surface-plasmon-resonance-enhanced cavity ring-down detection.

The cavity ring-down technique is used to probe the absolute optical response of the localized surface plasmon resonance (SPR) of a gold nanoparticle distribution to adsorption of trichloroethylene (TCE) and perchloroethylene (PCE) from the gas phase. Extended Mie theory for a coated sphere with a particle-size-dependent dielectric function is used to elucidate size-dispersion effects, the size-dependence of the SPR sensitivity to adsorption, and the kinetics of adsorption. An approximate Gaussian distribution of nanospheres with a mean diameter of 4.5 nm and a standard deviation of 1.1 nm, as determined by atomic force microscopy, is provided by the intrinsic granularity of an ultrathin, gold film, having a nominal thickness of approximately 0.18 nm. The cavity ring-down measurements employ a linear resonator with an intracavity flow cell, which is formed by a pair of ultrasmooth, fused-silica optical flats at Brewsters angle, where the Au film is present on a single flat. The total system intrinsic loss is dominated by the film extinction, while the angled flats alone contribute only approximately 5 x 10(-5)/flat to the total loss. Based on a relative ring-down time precision of 0.1% for ensembles averages of 25 laser shots from a pulsed optical parametric oscillator, the minimum detectable concentrations of PCE and TCE obtained by probing the SPR response are found to be 2 and 7 x 10(-8) mol/L, respectively, based on a 30 s integration time.

Experimental demonstration of phase determination in neutron reflectometry by variation of the surrounding media

Abstract We have recently shown that it is possible to unambiguously determine the real part of the complex reflection coefficient for a thin film structure by measuring the neutron specular reflectivities for the film in contact with two different backing or two different fronting media, thus simplifying the reference methodology of phase determination and making it more practical. Here we demonstrate the technique in two different experiments. In the first, one backing medium is air, the other, heavy water. In the second, sapphire and silicon serve as two different fronting (incident) media. In addition, we show how the two possible branches of the imaginary part of the reflection coefficient inferred by the real part, while not required for inverting the data to find the film structure, can be a sensitive diagnostic for indicating whether the film of interest is inhomogeneous on a transverse scale comparable to the neutron coherence length. This is especially important in avoiding misinterpretation of data which result from the incoherent average of reflectivities from large-scale heterogeneities.

Mechanism of formation of vesicle fused phospholipid monolayers on alkanethiol self-assembled monolayer supports.

We investigated the process of phospholipid vesicle fusion to a hydrophobic surface using electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The kinetics of the fusion of dimyristoyl phosphatidylcholine (DMPC) vesicles to an octadecanethiol (ODT) self-assembled monolayer (SAM) was followed with EIS and found to be slower than previously reported observations by surface plasmon resonance. AFM images of the DMPC layer taken at various stages of completion show they are uniform with topography similar to that of the bare ODT SAM. The AFM was used in contact mode to scrape away a portion of a completed DMPC monolayer and the hole was stable for several hours. Surprisingly, the hole remained stable even after the temperature of the system was raised to 38 degrees C, well above the gel transition temperature for DMPC of 23 degrees C. We were unable to scrape holes in the partially completed monolayers. We conclude that in the early stages of monolayer growth the DMPC molecules spread across the ODT surface after vesicle fusion rather than remaining in dense islands. However, when the DMPC monolayer is complete this mobility is lost and the DMPC will not spread into a vacant area.

Tertiary structure changes in albumin upon surface adsorption observed via fourier transform infrared spectroscopy.

A nondestructive Fourier transform infrared (FTIR) spectroscopy assay, amenable to exploring a wide range of proteins and polymers, is used to measure changes in the tertiary structure of bovine serum albumin (BSA) adsorbed to three surfaces: gold, polystyrene (PS), and poly(D,L-lactic acid) (PDLLA). Tertiary structural analysis is important because typical secondary structural analysis (FTIR and CD) is not always sensitive enough to distinguish between the sometimes subtle protein structural changes caused by adsorption. The polymers are spin-coated onto a gold surface, exposed to protein, and then immersed in a deuterated buffer solution to probe the proteins tertiary structure before the sample is removed from its aqueous environment. Infrared band intensities, related to the exchange of amide hydrogen for deuterium (HDX), as a function of the immersion time in deuterated buffer, are used to determine the extent of amide solvent exposure. Analysis of the results in terms of a single exponential decay shows that enough amides undergo a measurable amount of exchange in 60 min to quantify relative changes in BSA solvent exposure on different surfaces. In addition, substantial fractions undergo HDX at a rate too fast or too slow to be followed with our experimental protocol. The proportions of these quickly and slowly exchanging amide groups also provide information about relative changes in the BSA structure on different surfaces. Adsorption was found to increase the extent of HDX over that observed for BSA in solution, consistent with surface-induced unfolding and a loss of tertiary structure. Changes in HDX were found to be more sensitive to which surface was absorbing the protein than the typical FTIR secondary structural analysis obtained from fitting the amide I band. HDX was greatest for BSA adsorbed to the surface of PDLLA and least in the case of BSA adsorbed to gold, which indicates the greatest and least degree of unfolding, respectively.

Infrared techniques for quantifying protein structural stability.

Biopharmaceutical and biotechnology companies and regulatory agencies require novel methods to determine the structural stabilities of proteins and the integrity of protein-protein, protein-ligand, and protein-membrane interactions that can be applied to a variety of sample states and environments. Infrared spectroscopy is a favorable method for a number of reasons: it is adequately sensitive to minimal sample amounts and is not limited by the molecular weight of the sample; yields spectra that are simple to evaluate; does not require protein modifications, a special supporting matrix, or internal standard; and is applicable to soluble and membrane proteins. In this paper, we investigate the application of infrared spectroscopy to the quantification of protein structural stability by measuring the extent of amide hydrogen/deuterium exchange in buffers containing D(2)O for proteins in solution and interacting with ligands and lipid membranes. We report the thermodynamic stability of several protein preparations, including chick egg-white lysozyme, trypsin bound by benzamidine inhibitors, and cytochrome c interacting with lipid membranes of varying net-negative surface charge density. The results demonstrate that infrared spectroscopy can be used to compare protein stability as determined by amide hydrogen/deuterium exchange for a variety of cases.

International comparability in spectroscopic measurements of protein structure by circular dichroism: CCQM-P59.1

Circular dichroism (CD) is a spectroscopic technique that is widely used to obtain information about protein structure, and hence is an important tool with many applications, including the characterization of biopharmaceuticals. A previous inter-laboratory study, CCQM-P59, showed that there was a poor level of comparability between laboratories in CD spectroscopy. In a follow-up study reported here, we achieved our goal of demonstrating improved comparability and data quality, primarily by addressing the problems identified in the previous study, which included cell path-length measurement, instrument calibration and good practice in general. Multivariate analysis techniques (principal component analysis and soft independent modelling of class analogies) were shown to be useful in comparing large spectral data sets and in classifying spectra. However, our results also show that there is more work to be done to improve confidence in the technique as the discrepancies observed were partially due to systematic effects, which the statistical approaches do not consider. We therefore conclude that there is a need for an improved understanding of the uncertainties in CD measurement.

Infrared and Visible Absolute and Difference Spectra of Bacteriorhodopsin Photocycle Intermediates

We have used new kinetic fitting procedures to obtain infrared (IR) absolute spectra for intermediates of the main bacteriorhodopsin (bR) photocycle(s). The linear-algebra-based procedures of Hendler et al. (J. Phys. Chem. B, 105, 3319–3228 (2001) for obtaining clean absolute visible spectra of bR photocycle intermediates were adapted for use with IR data. This led to isolation, for the first time, of corresponding clean absolute IR spectra, including the separation of the M intermediate into its MF and MS components from parallel photocycles. This in turn permitted the computation of clean IR difference spectra between pairs of successive intermediates, allowing for the most rigorous analysis to date of changes occurring at each step of the photocycle. The statistical accuracy of the spectral calculation methods allows us to identify, with great confidence, new spectral features. One of these is a very strong differential IR band at 1650 cm−1 for the L intermediate at room temperature that is not present in analogous L spectra measured at cryogenic temperatures. This band, in one of the noisiest spectral regions, has not been identified in any previous time-resolved IR papers, although retrospectively it is apparent as one of the strongest L absorbance changes in their raw data, considered collectively. Additionally, our results are most consistent with Arg82 as the primary proton-release group (PRG), rather than a protonated water cluster or H-bonded grouping of carboxylic residues. Notably, the Arg82 deprotonation occurs exclusively in the MF pathway of the parallel cycles model of the photocycle.