Wolfgang Frey

Light scattering microscopy from monolayers and nanoparticles at the air/water interface

Abstract Light scattering microscopy (LSM) is introduced here as a versatile technique for the study of interfacial films at the air/water interface. Laser light scattered from the interface is collected by a microscope objective and imaged onto a CCD camera. LSM allows determination of the spatial distribution of submicron particles, phase transitions in two-dimensions, and defects. The power of LSM, especially if conducted simultaneously with fluorescence microscopy or Brewster angle microscopy (BAM), is illustrated in three examples. (a) Visualization of the spatial distribution of nanoscale particles with respect to monolayer phases: calcium oxalate crystals were grown underneath dipalmitoyl phosphatidyl choline (DPPC) and dimyristoyl phosphatidyl serine (DMPS) monolayers in the liquid expanded/liquid condensed (LE/LC) phase coexistence. The density of light scatterers was considerably higher in the respective LE than the LC phase, and a gradual migration of particles was observed towards the domain boundaries. A similar behavior was observed for nanocrystals injected underneath lipid monolayers. (b) Probing two-dimensional (2D) protein crystallization processes underneath functionalized lipid monolayers and the spatial distribution of crystal defects: streptavidin was crystallized as a model protein in 2D through coordination of exposed histidines on the protein surface to the monolayer-anchored metal chelator, Cu-DO-IDA. Vacancies were formed within the 2D protein crystals after injection of the soluble metal chelator EDTA, and the spatial distribution of vacancies was probed by LSM. (c) Detection of monolayer topographic transitions, corrugation and nanoscale budding: The phases of DPPC monolayers were studied under isothermal compression. New nanoscale topographic transitions are observed by LSM if DPPC is compressed into the liquid condensed (LC) state far below the collapse pressure.

Electron crystallographic analysis of two-dimensional streptavidin crystals coordinated to metal-chelated lipid monolayers.

Coordination of individual histidine residues located on a protein surface to metal-chelated lipid monolayers is a potentially general method for crystallizing proteins in two dimensions. It was shown recently by Brewster angle microscopy (BAM) that the model protein streptavidin binds via its surface histidines to Cu-DOIDA lipid monolayers, and aggregates into regularly shaped domains that have the appearance of crystals. We have used electron microscopy to confirm that the domains are indeed crystalline with lattice parameters similar to those of the same protein crystallized beneath biotinylated lipid monolayers. Although BAM demonstrates that the two-dimensional protein crystals grown via metal chelation are distinct from the biotin-bound crystals in both microscopic shape and thermodynamic behavior, the two crystal types show similar density projections and the same plane group symmetry.

Dynamics of two-dimensional protein crystallization at the air/water interface: streptavidin targetted to surfaces via high-affinity binding or metal coordination

Two approaches to target proteins to monolayers at the air/water interface are compared in order to investigate the influence of the surface binding mechanism on the formation of two-dimensional (2D) protein crystals. Surface binding of the model protein streptavidin is achieved by (a) high-affinity binding to a biotinylated monolayer, and (b) coordination of surface histidines to a metal chelated IDA-lipid monolayer. Streptavidin crystallizes in both cases. The crystallization process and the crystal shapes, however, are significantly different. In a comparative study utilizing Brewster angle microscopy, we gained quantitative information regarding both the critical surface density required for the formation of 2D crystals and the compressibility of the crystalline and noncrystalline phases. Possible influences of the protein orientation, protein-protein contacts, and multiple protein populations are discussed.

Binding and two-dimensional crystallization of streptavidin at the air/water interface via engineered Cu-IDA chelator lipids

Abstract Metal-chelating iminodiacetate (IDA) lipid monolayers at the air/water interface have been engineered to promote the 2D crystallization of proteins which display solvent-accessible histidines. The physical properties of IDA lipid monolayers have been specifically tailored to allow the binding and 2D assembly of added proteins. Fluorescently labeled IDA lipid monolayers allow us to monitor the formation of the protein crystals optically. Here we review our efforts in the interfacial crystallization of streptavidin as monitored using fluorescence, Brewster angle and transmission electron microscopy and discuss future challenges in this area.

Two-dimensional crystallization of streptavidin: in pursuit of the molecular origins of structure, morphology, and thermodynamics

The streptavidin two-dimensional (2D) crystallization model has served as a paradigm for molecular self-assembly at interfaces. We have developed quantitative Brewster angle microscopy for the in situ measurement of spatially resolved relative protein surface densities. This allows investigation of both the thermodynamics and morphologies of 2D crystal growth. For crystal structure analysis, we employ TEM on grown crystals transferred to solid substrates. Comparison of results between commercially available streptavidin, recombinant streptavidin, and site-directed streptavidin mutants has provided insight into the protein protein and protein-lipid interactions that underlie 2D crystallization.

Polymer nanogels grafted from nanopatterned surfaces studied by AFM force spectroscopy.

Nanopatterned cross-linked polymers are important for applications with controlled mechanical properties. Grafted linear and cross-linked polydimethylacrylamide gels on micro- and nanopatterns were created using iniferter-driven quasi-living radical polymerization combined with conventional photolithography and nanosphere lithography. Micropatterned linear polymers reproduce the expected scaling behavior at moderate grafting density. The addition of cross-linker to the polymerization solution leads to an increased tendency of early termination as determined by AFM force spectroscopy. Similarly, nanopatterned linear polymers show reduced thickness in agreement with the expected scaling relationship for nanoisland grafts that have reduced lateral confinement. The addition of cross-linker reintroduces some of the lateral confinement for the length of polymers reported here. The mechanical properties of both the micro- and nanopatterned linear as well as cross-linked polymers were analyzed using an algorithm to objectively determine the contact point in AFM force spectroscopy and two independent Hertz-based analysis approaches. The obtained Youngs moduli are close to those expected for homogeneous thick polymer films and are independent of pattern size. Our results demonstrate that polymeric nanopillars with controlled elastic modulus can be fabricated using irreversible cross-linkers. They also highlight some of the factors that must be considered for successful fabrication of grafted nanopillars of defined mechanical and structural properties.