the liquid interface

Liquid interfaces govern many biological mechanisms, from the scale of a cell membrane to that of the marine microlayer. At liquid interfaces, transport and control of chemical species occurs; organized molecular structures provide critical physical and chemical functions.

The liquid interface also provides a flat organizational template at which structural measurements can be combined with other experimental techniques to learn about important biological and biomimetic systems.

Here we describe synchrotron x-ray scattering methods as applied to liquid surfaces, and a selection of recent (unfinished!) experiments designed to shed light on biological systems.

surface scattering geometry

At any interface between two media (such as water and air), differences in electron density will reflect and refract x-rays, resulting in an interference pattern. Detecting the scattered intensity as a function of angle allows the real-space structure to be modeled. Both surface-normal and in-plane structures can be obtained.

Reflection from a perfectly flat plane follows the Fresnel law of optics. Additional density modulations at the surface, caused by adsorbed molecules, roughness, or concentration gradients, are determined from the x-ray reflectivity.

To study structural correlations in the layer plane, grazing incidence diffraction is used. When the x-ray angle of incidence a is less than the critical angle for total external reflection, only the near-surface region is illuminated. When molecules order on a liquid surface, Bragg peaks appear at characteristic scattering angles related to in-plane d-spacings: l/d = 2sin(2q/2). Structure along the surface normal, for example of amphiphiles with tilted tails, is detected as a function of b. The related technique of grazing incidence small-angle scattering is sensitive to order on larger length scales.

Langmuir trough techniques

Various conditions may be established in the liquid system, including the atmosphere, temperature, and solution chemistry. A surfactant film may act as an artificial membrane for embedded biomolecules, or instead as a charged template for nucleation.

The Langmuir trough adds a particular measure of control to a liquid surface/surfactant system: measurement and modification of the surface pressure by means of a movable barrier and Wilhelmy balance. The pressure-area phase behavior of amphiphiles on liquids is related to structural phase transitions, which may affect the interactions of species at the interface.

The experiments described below were conducted at NSLS Beamline X22B and APS Beamline 9-ID (CMC-CAT). Brookhaven and Argonne National Labs are supported by the USDOE. BNL's Center for Functional Nanomaterials provides access to liquid surface scattering beamlines. Visit www.cfn.bnl.gov or contact Elaine DiMasi (dimasi@bnl.gov).

motivation

Resonance reflectivity, where the x-ray energy is tuned to an absorption edge to obtain elemental contrast, has been used to locate heavy atoms at discrete sites within the structure of protein films. Similarly, it should be possible to determine the separation between metallo-porphyrin prosthetic groups (eg. heme) in an electron-transfer chain, or locate the binding site for small molecules, such as the general anesthetic halothane (F3C-CClBrH). This requires control of the protein orientation, which can be achieved by spreading monolayers at the air/water interface and manipulating the protein/lipid ratio, area/molecule and applied surface pressure. Our objective is to develop the technique of resonant liquid surface reflectivity to obtain structural information in these systems. The CMC CAT Liquid Surface Spectrometer at Argonne National Laboratory allows energy tunability without affecting spectrometer alignment, and is ideal for these studies.

results

As a test case, we investigated monolayers spread from stearic acid and from 2-bromo-stearic acid. The energy dependence of the structure factor of Bromine results in different reflectivity from the monolayer at energies at and near the K-absorption edge.

The measured reflectivity curves exhibit a reproducible energy dependence only for the Br-monolayer, with no effect in the control stearic acid monolayer. Films remained stable and the correct x-ray energy dependence was observed. Analysis is underway!

Acknowledgments: NIH (U Penn, GM55876); DOE (NSLS, APS).

motivation

Photosynthesis, in higher plants, algae and cyanobacteria, takes place in the thylakoid membranes, the internal membranes of chloroplasts and cyanobacteria. In chloroplasts, these membranes, a unique assembly of protein, pigment and lipid molecules, accommodate all light-harvesting and energy transducing functions. The reaction centers are supplied with energy by light harvesting antenna complexes. In granal chloroplasts, the main chlorophyll a/b light harvesting complex LHCII accounts for about half of the protein and Chl content of the thylakoid membranes, and thus it is the most abundant membrane protein in the biosphere. These proteins, in addition to their primary, light harvesting function, also act as structure proteins. Recent structural data indicate that different lipids participate in specific lipid-protein interactions in reaction center and light harvesting complexes. However, their roles are still largely unknown.

results

To study the structure of the protein in a controlled environment with a similar chemical environment to that which the protein might experience in the membrane, we investigated monolayers of the LHCII complex and of associated lipids, assembled on water surfaces. The LHCII behaved as a slightly unstable surfactant. Pressure-area isotherms consistently exhibited an inflection point at surface pressures of about 25 mN/m, but with variable low-pressure slopes and small extrapolated molecular areas indicating that significant amounts of material were lost from the surface.

Reflectivities show dense regions 10 Å from the interface, probably associated lipid headgroups. Lipids extracted separately exhibited uninflected pressure-area isotherms, and could be stabilized up to 50 mN/m. Model profiles clearly show the headgroup and extended hydrocarbon tails, which are somewhat less extended at lower surface pressures.

Neither light-induced structural changes nor in-plane ordering were observed. We will address these questions in future studies, as they bear directly upon the protein complex's function.

motivation

Biogenic minerals often rival engineered materials, having specific hierarchical structures which lead to finely tuned physical and chemical properties. To achieve comparable control over submicron architecture and materials properties remains a significant motivation for materials science. To control biomineralization, the organism must collect the constituent species and control the solution chemistry (and thus the kinetics of the reaction). Soluble macromolecules and proteins, as well as insoluble organic material, are generally present. Whether the organic matter serves as a structural scaffold, or as an atomic-scale template for crystal nucleation, has been a matter of ongoing debate. We have applied in-situ x-ray scattering techniques to study mineralization of calcium carbonates at monolayer films, where the mineral orientation and crystallinity can be assessed simultaneously with a determination of the template structure. (NSLS Beamline X22B.)

results

Using reflectivity and in-plane scattering measurements, we have determined that no structural registry occurs between fatty acid monolayers and CaCO₃ crystals that precipitate against the film. Instead, polytype selectivity between metastable vaterite and stable calcite minerals is determined by competition between kinetic factors (gas escape) and charge gradients (cations bound to the monolayer film). Acidic polypeptides, such as poly(acrylic acid), have particularly dramatic effects on mineralization, leading to amorphous precursor phases.

Time resolved x-ray measurements reveal that the polymer removes cations from the monolayer, affecting the relative stability of CaCO₃ phases. This chelating mechanism may be one way in which proteins control biogenic mineralization.