Luka Pocivavsek

Stress and Fold Localization in Thin Elastic Membranes

Thin elastic membranes supported on a much softer elastic solid or a fluid deviate from their flat geometries upon compression. We demonstrate that periodic wrinkling is only one possible solution for such strained membranes. Folds, which involve highly localized curvature, appear whenever the membrane is compressed beyond a third of its initial wrinkle wavelength. Eventually the surface transforms into a symmetry-broken state with flat regions of membrane coexisting with locally folded points, reminiscent of a crumpled, unsupported membrane. We provide general scaling laws for the wrinkled and folded states and proved the transition with numerical and experimental supported membranes. Our work provides insight into the interfacial stability of such diverse systems as biological membranes such as lung surfactant and nanoparticle thin films.

Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

BACKGROUND Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained. RESULTS We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.

Geometric tools for complex interfaces: from lung surfactant to the mussel byssus

Interfaces are ubiquitous in nature and absolutely key for life as illustrated by such complex interfaces as the cell membrane and the endothelial and epithelial linings of tissues. The mechanical properties of these interfaces play an important role in their biological functions. In this highlight, we describe our recent work (Pocivavsek et al., Science, 2008, 320, 912) using geometry as a tool for studying the behavior of complex interfaces. General scaling laws emerging from studying the shape of elastic interfaces can in turn be used in their characterization. Interfacial wrinkling is a well known phenomenon, however, the geometric patterns seen at biological interfaces are often far from idealized sinusoidal wrinkles. We show how more complex and non-linear patterns naturally emerge from wrinkles and how material properties can be extracted from these non-linear geometries.

Wilhelmy plate artifacts in elastic monolayers

A recent article [L. Pocivavsek et al., Soft Matter4, 2019 (2008)] by some of us pointed out difficulties in interpreting Wilhelmy plate measurements on elastic Langmuir monolayers that support anisotropic stress. Using a simplified geometry it showed conditions in which the Wilhelmy plate measures significantly different stress from the ambient stress. We correct a serious error in this analysis and strengthen its conclusion, showing that the Wilhelmy stress and the ambient stress can have opposite signs.

Analysis of biosurfaces by neutron reflectometry: From simple to complex interfaces

Because of its high sensitivity for light elements and the scattering contrast manipulation via isotopic substitutions, neutron reflectometry (NR) is an excellent tool for studying the structure of soft-condensed material. These materials include model biophysical systems as well as in situ living tissue at the solid-liquid interface. The penetrability of neutrons makes NR suitable for probing thin films with thicknesses of 5-5000 Å at various buried, for example, solid-liquid, interfaces [J. Daillant and A. Gibaud, Lect. Notes Phys. 770, 133 (2009); G. Fragneto-Cusani, J. Phys.: Condens. Matter 13, 4973 (2001); J. Penfold, Curr. Opin. Colloid Interface Sci. 7, 139 (2002)]. Over the past two decades, NR has evolved to become a key tool in the characterization of biological and biomimetic thin films. In the current report, the authors would like to highlight some of our recent accomplishments in utilizing NR to study highly complex systems, including in-situ experiments. Such studies will result in a much better understanding of complex biological problems, have significant medical impact by suggesting innovative treatment, and advance the development of highly functionalized biomimetic materials.

Understanding dynamic changes in live cell adhesion with neutron reflectometry.

Neutron reflectometry (NR) was used to examine various live cells adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutron reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell - surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies.

Initiating a structural study of 2-keto-3-deoxy-6- phosphogluconate aldolase from Escherichia coli

2-Keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase, E.C. 4.1. 2.14) is a member of the pyruvate/phosphoenolpyruvate aldolase family. It is also a synthetically useful enzyme, capable of catalyzing the stereoselective aldol addition of pyruvate to a range of unnatural electrophilic substrates. The recombinant protein was purified by a two-step HPLC protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated the protein to be monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method, with PEG 6K as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.26 A on station PX9.6 at the Daresbury synchrotron. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.2, b = 77.9, c = 146.8 A.

Confined disclinations: exterior versus material constraints in developable thin elastic sheets.

We examine the shape change of a thin disk with an inserted wedge of material when it is pushed against a plane, using analytical, numerical, and experimental methods. Such sheets occur in packaging, surgery, and nanotechnology. We approximate the sheet as having vanishing strain, so that it takes a conical form in which straight generators converge to a disclination singularity. Then, its shape is that which minimizes elastic bending energy alone. Real sheets are expected to approach this limiting shape as their thickness approaches zero. The planar constraint forces a sector of the sheet to buckle into the third dimension. We find that the unbuckled sector is precisely semicircular, independent of the angle δ of the inserted wedge. We generalize the analysis to include conical as well as planar constraints and thereby establish a law of corresponding states for shallow cones of slope ε and thin wedges. In this regime, the single parameter δ/ε^{2} determines the shape. We discuss the singular limit in which the cone becomes a plane, and the unexpected slow convergence to the semicircular buckling observed in real sheets.

In situ Monitoring of Structural Changes in Model Membranes upon Cholesterol Depletion via X-ray Diffraction

The importance of cholesterol in the molecular structure and organization of cell membranes is a topic of great research interest. It has been hypothesized that the lateral heterogeneity of cell membranes arises from the dynamic self-assembly of cholesterol enriched nanodomains. In order to elucidate the fundamental molecular interactions involved in the assembly of these nanodomains, binary lipid monolayers of dimyristoylphosphatidylethanolamine (DMPE) and dihydrocholesterol (DChol) were studied as model systems and probed using grazing incidence x-ray diffraction (GIXD). Mixed DMPE/DChol systems were shown to exhibit short-ranged lateral ordering consistent with previous data for a lipidic alloy of egg sphingomyelin and DChol that obeys Vegards law [Phys. Rev. Lett 2009, 103, 028103]. In the presence of β-cyclodextrin (CD), DChol was selectively removed from the membrane. GIXD was used to monitor the changes of lipid ordering during CD mediated desorption of DChol to the subphase. The chemical of amount of CD to DChol was greater than a factor of 1000 and complete DChol depletion was expected. However, it was observed that a significant amount of DChol remains in the membrane during the experimental time frame of a couple of hours and this resistance to CD transfer could be due to the stability of condensed complexes formed between DMPE and DChol.