In this presentation we review our recent work using x-ray reflectivity to determine the configuration of membrane-bound proteins. The reflectivity data is analyzed in terms of the known crystallographic structure of proteins and a slab model representing the lipid layer to yield an electron density profile of the lipid/protein system. Our recent modified analysis methodology for the lipid/protein system is concisely described in this report. In addition, some results of the configuration of the membrane-bound proteins cPLA2α-C2, p40phox-PX, and PKCα-C2 are highlighted.

An understanding of signal transduction mechanisms is vital to investigate the causes of diseases. The C2 domain is a conserved protein signaling motif and membrane-targeting domain widely found in signaling proteins. In this work, we study the interaction of the C2 domain of protein kinase Cα (PKCα) with a lipid monolayer of a mixture of SOPC (1-stearoyl-2-oleyl-sn-glycero-3-phosphocholine) and SOPS (1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine). Recent results from crystallography and EPR studies indicate that PKCα-C2 is likely to orient parallel to the membrane. In this work, we use x-ray reflectivity to directly determine that the PKCα-C2 domain is perpendicular to the membrane. Our new analysis method allows us to test all orientations and demonstrates that our data is inconsistent with the parallel orientation. To carry out this experiment, the PKCα-C2 was injected into the subphase under an SOPC/SOPS (7:3) mixture supported on a buffered aqueous solution. X-ray reflectivity was used to determine the orientation and penetration depth of PKCα-C2 bound to the SOPC/SOPS monolayer. The reflectivity is analyzed in terms of the known crystallographic structure of PKCα-C2 and a slab model that represents the lipid layer, yielding an electron density profile of the lipid layer and bound C2 domain. The orientation of PC/PS-bound PKCα-C2 is described by two angles, θ = 35° and φ = 210°, and the domain penetrates 7.6 Å into the lipid layer. The structure that we determined is consistent with many observations from mutational studies. The perpendicular model further suggests how PKCα-C2 interacts with other lipid components such as phosphatidylinositol, other domains within PKC such as the C1 domain, and the receptor for activated C-kinase.

Gold nanostars (NS) are emerging as a versatile tool in surface-enhanced Raman scattering (SERS) applications because of their wide localized surface plasmon resonance (LSPR) tunability, simple synthesis procedure, and high SERS enhancement. These particles are commonly used in solutions with a stabilizing coating shell (e.g., thiolated molecules or silver shell). However, coatings cannot be used for the fabrication of SERS substrates as the NS have to interact with the substrate planar surface. Without coating, NS have been observed to change over time, leading to a hypochromic shift of the LSPR. To understand this shift, we synthesized surfactant-free gold NS with different spike morphologies and investigated their reshaping morphology and kinetics. Using TEM, the NS sharp spike features were observed to reshape over time. The kinetics of this process were analyzed and determined by monitoring the LSPR, which was observed to follow an exponential decay over time. We used an empirical fit for the LSPR-shift data as a function of time, which permits to predict the LSPR at a specific time based only on the initial LSPR (independently of the initial spike morphology). We show the effect of the LSPR on the SERS signal for the NS and how the SERS signal correlated to our prediction. Finally, we evaluated our approach by fabricating SERS substrates with immobilized NS and collecting the reflectance spectra. We were able to predict the substrate LSPR and aim for an optimal LSPR with an average 3% deviation. These new insights on NS reshaping can permit the fabrication of NS-based substrates with desirable optical/plasmonic properties.

When partially polymerized membranes wrinkle they exhibit a passage from a conventional buckling (due to an instability caused by chiral symmetry breaking) at low polymerization to a local roughening (due to a frustration in the local packing of the chiral molecules composing the membrane) as a function of the polymerization of the lipids aliphatic tails. This transition was found to be non-universal and here we used neutron scattering to elucidate that this behavior is due to the onset of stretching in the membrane accompanied by a bilayer thickness variation. Close to the percolation limit this deformation is plastic similar to mutated lysozymes. We draw an analogy between this transition and echinocytes in red blood cells.

X-ray reflectivity was used to study the interaction of the PX domain of p40(phox) protein (p40(phox)-PX) with a Langmuir monolayer of a mixture of SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), SOPS (1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine), and DPPtdIns(3)P (1,2-dipalmitoylphosphatidylinositol 3-phosphate) lipids supported on a buffered aqueous solution. The reflectivity is analyzed in terms of the known crystallographic structure of the p40(phox)-PX domain and a slab model that represents the lipid layer, yielding an electron density profile of the lipid layer and bound PX domains. This analysis determines the angular orientation and penetration depth of the p40(phox)-PX domain bound to the SOPC/SOPS/DPPtdIns(3)P monolayer. The best fit orientation is characterized by the following angles: theta = 30 +/- 10 degrees and phi = 140 +/- 30 degrees. These angles describe rotations, about axes in a coordinate system fixed to the domain, that are required to orient the domain with respect to the lipid layer at the interface. The protein penetrated into the lipid layer by 9 +/- 2 A, indicating that the protein penetrated into the headgroup region, but not deeply into the hydrocarbon region of the monolayer. In this analysis, polar Tyr(94) and hydrophobic Val(95) penetrated deepest into the lipid monolayer. The backbone of these residues was approximately 5 A above the headgroup-buffer interface, i.e., at the level of the SOPC/SOPS lipid phosphates. Positively charged Lys(92) and Lys(98) were also near the SOPC/SOPS lipid phosphates. This position of the protein allows for a favorable electrostatic contribution to binding.

Front Cover: TEM images (ultrathin section stained with ruthenium oxide vapor) show essential features of the self‐assembled structure of the weakly segregated bulk blends of d8PS‐block‐PMMA copolymer and PS homopolymer with similar degrees of polymerization. An example of “dry brush” micromorphology (a compact lamellar domain of the copolymer surrounded by homopolymer matrix) is given in the inset. Further details can be found in the Full Paper by J. Holoubek,* J. Baldrian, F. Lednický, Š. Mlkov, and J. Lal on page 1834.

Mean field theories of ion distributions, such as the Gouy-Chapman theory that describes the distribution near a charged planar surface, ignore the molecular-scale structure in the liquid solution. The predictions of the Gouy-Chapman theory vary substantially from our x-ray reflectivity measurements of the interface between two electrolyte solutions. Molecular dynamics simulations, which include the liquid structure, were used to calculate the potential of mean force on a single ion. We used this potential of mean force in a generalized Poisson-Boltzmann equation to predict the full ion distributions. These distributions agree with our measurements without any adjustable parameters.

Synchrotron X-ray reflectivity is used to study ion distributions at the liquid–liquid interface between a nitrobenzene solution of tetrabutylammonium tetraphenylborate (TBATPB) and a water solution of tetrabutylammonium bromide (TBABr). The concentration of TBABr is varied to alter the ion distribution. Our X-ray measurements are inconsistent with several commonly used theories of ion distributions, including Gouy–Chapman, modified Verwey–Niessen, and the MPB5 version of the Poisson–Boltzmann equation. These structural measurements are described well by ion distributions predicted by a version of the Poisson–Boltzmann equation that explicitly includes a free energy profile for ion transfer across the interface when this profile is described by a simple analytic form or by a potential of mean force from molecular dynamics simulations. These X-ray measurements from the liquid–liquid interface provide evidence for the importance of interfacial liquid structure in determining interfacial ion distributions.

The study describes the investigation of the microphase-separated morphology of a block copolymer A-block-B with the parent homopolymer A, where A is polystyrene and A-block-B is poly(perdeuterated styrene)-blockpoly(methyl methacrylate) (dPS-block-PMMA). The microdomain morphology and phase behavior in blends of dPS-block-PNIMA with polystyrene of (M) over bar (W) =8000 and 35000 were investigated. Binary blends of the diblock copolymer and homopolymers were prepared with various amounts of homopolymers. The criterion for "wet and dry brush" taking into account different solubilization of homopolymer has been applied to explain the changes in microdomain morphology during the self-assembling process.

Synchrotron X-ray reflectivity is used to study the electron density as a function of depth through the bulk nitrobenzene-water interface at four different temperatures. The measured interfacial width differs from the predictions of capillary wave theory with a progressively smaller deviation as the temperature is raised. Computer simulations suggest the presence of both molecular layering and dipole ordering parallel to the interface. Either layering or a bending rigidity, that can result from dipole ordering, can explain these measurements.

X-ray reflectivity is used to study the interaction of C2 domains of cytosolic phospholipase A2 (cPLA2α-C2) with a Langmuir monolayer of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) supported on a buffered aqueous solution containing Ca2+. The reflectivity is analyzed in terms of the known crystallographic structure of cPLA2α-C2 domains and a slab model representing the lipid layer to yield an electron density profile of the lipid layer and bound C2 domains. This new method of analysis determines the angular orientation and penetration depth of the cPLA2α-C2 domains bound to the SOPC monolayer, information not available from the standard slab model analysis of x-ray reflectivity. The best-fit orientation places the protein-bound Ca2+ ions within 1Å of the lipid phosphate group (with an accuracy of ±3Å). Hydrophobic residues of the calcium-binding loops CBL1 and CBL3 penetrate deepest into the lipid layer, with a 2Å penetration into the tailgroup region. X-ray measurements with and without the C2 domain indicate that there is a loss of electrons in the headgroup region of the lipid monolayer upon binding of the domains. We suggest that this is due to a loss of water molecules bound to the headgroup. Control experiments with a non-calcium buffer and with domain mutants confirm that the cPLA2α-C2 binding to the SOPC monolayer is Ca2+-dependent and that the hydrophobic residues in the calcium-binding loops are critical for membrane binding. These results indicate that an entropic component (due to water loss) as well as electrostatic and hydrophobic interactions contributes to the binding mechanism.

Synchrotron X-ray reflectivity is used to study the electron density as a function of depth through the bulk water/2-heptanone interface. The measured interfacial width of 7.0±0.2Å is comparable to the value calculated from capillary wave theory (7.3Å) using the measured interfacial tension of 12.6mN/m. This result is consistent with capillary wave theory and molecular dynamics simulations that describe a molecularly sharp interface roughened by thermal fluctuations.

We demonstrate the use of X-ray reflectivity to probe the electron density profile normal to the interface between two polar liquids. Measurements of the interfacial width at the neat nitrobenzene/water and the neat water/2-heptanone interfaces are presented. These widths are consistent with predictions from capillary wave theory that describe thermal interfacial fluctuations determined by the tension and bending rigidity of the interface. Variation of the temperature of the water/nitrobenzene interface from 25 degrees C to 55 degrees C indicates that the role of the bending rigidity decreases with increasing temperature. X-ray reflectivity measurements of the electrified interface between an aqueous solution of BaCl2 and a nitrobenzene solution of TBATPB demonstrate the sensitivity of these measurements to the electrolyte distribution at the interface. A preliminary analysis of these data illustrates the inadequacy of the simplest, classical Gouy-Chapman theory of the electrolyte distribution.