Probing gigahertz coherent acoustic phonons in TiO2 mesoporous thin films.

Ultrahigh-frequency acoustic-phonon resonators usually require atomically flat interfaces to avoid phonon scattering and dephasing, leading to expensive fabrication processes, such as molecular beam epitaxy. Mesoporous thin films are based on inexpensive wet chemical fabrication techniques that lead to relatively flat interfaces regardless the presence of nanopores. Here, we report mesoporous titanium dioxide-based acoustic resonators with resonances up to 90 GHz, and quality factors from 3 to 7. Numerical simulations show a good agreement with the picosecond ultrasonics experiments. We also numerically study the effect of changes in the speed of sound on the performance of the resonator. This change could be induced by liquid infiltration into the mesopores. Our findings constitute the first step towards the engineering of building blocks based on mesoporous thin films for reconfigurable optoacoustic sensors.

Three-Dimensional Electrical Control of the Excitonic Fine Structure for a Quantum Dot in a Cavity.

The excitonic fine structure plays a key role for the quantum light generated by semiconductor quantum dots, both for entangled photon pairs and single photons. Controlling the excitonic fine structure has been demonstrated using electric, magnetic, or strain fields, but not for quantum dots in optical cavities, a key requirement to obtain high source efficiency and near-unity photon indistinguishability. Here, we demonstrate the control of the fine structure splitting for quantum dots embedded in micropillar cavities. We propose and implement a scheme based on remote electrical contacts connected to the pillar cavity through narrow ridges. Numerical simulations show that such a geometry allows for a three-dimensional control of the electrical field. We experimentally demonstrate tuning and reproducible canceling of the fine structure, a crucial step for the reproducibility of quantum light source technology.

Fiber-based angular filtering for high-resolution Brillouin spectroscopy in the 20-300†GHz frequency range.

Brillouin spectroscopy emerges as a promising non-invasive tool for nanoscale imaging and sensing. One-dimensional semiconductor superlattice structures are eminently used for selectively enhancing the generation or detection of phonons at few GHz. While commercially available Brillouin spectrometers provide high-resolution spectra, they consist of complex experimental techniques and are not suitable for semiconductor cavities operating at a wide range of optical wavelengths. We develop a pragmatic experimental approach for conventional Brillouin spectroscopy that can integrate a widely tunable excitation-source. Our setup combines a fibered-based angular filtering and a spectral filtering based on a rotating single etalon and a double grating spectrometer for sequential reconstruction of Brillouin spectra. This configuration allows probing confined acoustic phonon modes in the 20-300 GHz frequency range with excellent laser rejection and high spectral resolution. Remarkably, our scheme based on the excitation and collection of the enhanced Brillouin scattering signals through the optical cavity allows for better angular filtering with decreasing phonon frequency. It can be implemented for the study of cavity optomechanics and stimulated Brillouin scattering over broadband optical and acoustic frequency ranges.

Sequential generation of linear cluster states from a single photon emitter.

Light states composed of multiple entangled photons-such as cluster states-are essential for developing and scaling-up quantum computing networks. Photonic cluster states can be obtained from single-photon sources and entangling gates, but so far this has only been done with probabilistic sources constrained to intrinsically low efficiencies, and an increasing hardware overhead. Here, we report the resource-efficient generation of polarization-encoded, individually-addressable photons in linear cluster states occupying a single spatial mode. We employ a single entangling-gate in a fiber loop configuration to sequentially entangle an ever-growing stream of photons originating from the currently most efficient single-photon source technology-a semiconductor quantum dot. With this apparatus, we demonstrate the generation of linear cluster states up to four photons in a single-mode fiber. The reported architecture can be programmed for linear-cluster states of any number of photons, that are required for photonic one-way quantum computing schemes.

Optomechanical properties of GaAs/AlAs micropillar resonators operating in the 18 GHz range.

Recent experiments demonstrated that GaAs/AlAs based micropillar cavities are promising systems for quantum optomechanics, allowing the simultaneous three-dimensional confinement of near-infrared photons and acoustic phonons in the 18-100 GHz range. Here, we investigate through numerical simulations the optomechanical properties of this new platform. We evidence how the Poisson's ratio and semiconductor/vacuum boundary conditions lead to very distinct features in the mechanical and optical three-dimensional confinement. We find a strong dependence of the mechanical quality factor and strain distribution on the micropillar radius, in great contrast to what is predicted and observed in the optical domain. The derived optomechanical coupling constants g0 reach ultra-large values in the 106 rad/s range.

Conformational heterogeneity and spin-labeled -SH groups: pulsed EPR of Na,K-ATPase.

Membranous Na,K-ATPase from shark salt gland and from pig kidney was spin-labeled on class I -SH groups in the presence of glycerol, or on class II -SH groups in the absence of glycerol. The class I-labeled preparations retain full enzymatic activity, whereas the class II-labeled preparations are at least partially inactivated. This provides an excellent testbed on which to demonstrate how advanced electron paramagnetic resonance (EPR) can provide novel information on specific residues in unique environments in a complex, membrane-bound transport system. The polarity of the environment, and the librational dynamics and conformational exchange, of the spin-labeled groups were studied with pulsed EPR by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy, respectively. 2H-ESEEM spectra of membranes dispersed in D2O reveal that class I groups of the shark enzyme are more exposed to water than are those of the pig enzyme or class II groups of either species, consistent with the more superficial membrane location in the former case. Spin-echo decay curves indicate conformational heterogeneity at low temperatures (<150 K), but a more homogeneous conformational state at higher temperatures that is characterized by a single phase-memory T2M relaxation time. Conventional EPR lineshapes also demonstrate conformational microheterogeneity at low temperatures: the inhomogeneously broadened lines narrow progressively with increasing temperature reaching an almost pure Lorentzian line shape at temperatures of ca. 220 K and above. The inhomogeneous broadening at low temperature is well described by a Gaussian distribution of Lorentzian lines. ED spectra as a function of echo-delay time demonstrate the onset of rapid librational motions of appreciable amplitude, and slower conformational exchange, at temperatures above 220 K. These motions could drive transitions between the different conformational substates, which are frozen in at lower temperatures but contribute to the pathways between the principal enzymatic intermediates at higher temperatures.

Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR.

Several biologically active derivatives of the cardiotonic steroid ouabain have been made containing NMR isotopes ((13)C, (2)H, and (19)F) in the rhamnose sugar and steroid moieties, and examined at the digitalis receptor site of renal Na(+)/K(+)-ATPase by a combination of solid-state NMR methods. Deuterium NMR spectra of (2)H-labeled inhibitors revealed that the sugar group was only loosely associated with the binding site, whereas the steroid group was more constrained, probably because of hydrogen bonding to residues around the K(+)-channel region. Crosspolarization magic-angle spinning NMR showed that chemical shifts of inhibitors (13)C-labeled in the sugar group moved downfield by 0.5 ppm after binding to the digitalis site, suggesting that the sugar was close to aromatic side groups. A (19)F, (13)C- rotational-echo double-resonance NMR strategy was used to determine the structure of an inhibitor in the digitalis receptor site, and it showed that the ouabain derivatives adopt a conformation in which the sugar extends out of the plane of the steroid ring system. The combined structural and dynamic information favors a model for inhibition in which the ouabain analogues lie across the surface of the Na(+)/K(+)-ATPase alpha-subunit with the sugar group facing away from the surface of the membrane but free to move into contact with one or more aromatic residues.

Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation.

Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.

Microsecond motions of the lipids associated with trypsinized Na,K-ATPase membranes. Progressive saturation spin-label electron spin resonance studies.

The microsecond motions of spin-labeled lipids associated with the Na(+)/K(+)-transporting ATP hydrolase (Na,K-ATPase) in native and tryptically shaved membranes from Squalus acanthias have been studied by progressive saturation electron spin resonance (ESR). This includes both the segmental mobility of the lipid chains and the exchange dynamics of the lipids interacting directly with the protein. The lipids at the protein interface display a temperature-dependent chain mobility on the submicrosecond time scale. Exchange of these lipids with those in the bulk bilayer regions of the membrane takes place on the time scale of the nitroxide spin-lattice relaxation, i.e., in the microsecond regime. The off-rates for exchange directly reflect the specificity of ionized fatty acids relative to protonated fatty acids for interaction with the Na,K-ATPase. These essential features of the lipid dynamics at the intramembranous protein surface, namely, a temperature-dependent exchange on the microsecond time scale that reflects the lipid selectivity, are preserved on removing the extramembranous parts of the Na,K-ATPase by extensive trypsinization.

Selectivity of lipid-protein interactions with trypsinized Na, K-ATPase studied by spin-label EPR.

The selectivity of the lipid-protein interactions in trypsinised Na, K-ATPase membranes from Squalus acanthias has been determined by using EPR spectroscopy with different lipid probes spin-labelled on the 14-C atom of the fatty acid chain. From measurements at low ionic strength and different pH values, the pattern of selectivity is: (stearic acid)->(phosphatidylserine)->(stearic acid)0>(phosphatidylcholine)+/-, where superscripts indicate the formal electrostatic charge on the lipid headgroup. This is in the same order as that determined with native Na,K-ATPase membranes [M. Esmann, D. Marsh, Biochemistry 24 (1985) 3572-3578]. The selectivity for phosphatidylserine is independent of pH, over the range pH 6.0-9. 0, as found also for native membranes. For membranes trypsinised in the presence of Rb+ ions, and in the presence of Na+ (which allows more extensive proteolysis), the relative association constants, Kr, of all lipids are the same as for control membranes, with the exception of ionised (stearic acid)- that shows the highest specificity. Therefore, both the stoichiometry and the principal determinants of the specificity of lipid-protein interaction are preserved on extensive trypsinisation of Na,K-ATPase membranes. This has implications for the location and arrangement of those amino acid side chains that determine the lipid selectivity of the native Na,K-ATPase.

Characterization of the secondary structure and assembly of the transmembrane domains of trypsinized Na,K-ATPase by Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has been used to compare native Na,K-ATPase-containing membranes with those trypsinized in the presence of either Rb+ or Na+ ions to remove the extramembranous parts of the protein. The protein secondary structure content deduced from the amide I band is approximately 30-35% alpha-helix, 37-40% beta-structure, and 13-15% random coil for native membranes from shark rectal gland and from pig kidney, in both the Na- and K-forms. Trypsinization in either Rb+ (a K+ congener) or Na+ removes approximately 35% of the amide I band intensity of native membranes from shark rectal gland. The protein secondary structural content of the trypsinized membranes lies in the range of approximately 23-32% alpha-helix, 37-46% beta-structure, and 12-18% random coil for the shark and kidney enzymes. The distribution of intensity between the bands corresponding to protonated and deuterium-exchanged alpha-helices, and between the component bands attributed to beta-structure, changes considerably on trypsinization, in the direction of a greater proportion of protonated alpha-helix and a broader range of frequencies for beta-structure. The kinetics of deuteration of the slowly exchanging population of protein amide groups is also changed on trypsinization. The mean rate constant for deuteration of trypsinized membranes is approximately half that for native membranes, whereas the proportion of amides contributing to this population increases on trypsinization. The temperature dependence of the amide I band in the Fourier transform infrared spectra indicates that the onset of thermal denaturation occurs at 58 degrees C for native membranes (in either Na+ or K+) and for membranes trypsinized in Rb+, but the major denaturation event for membranes trypsinized in Na+ occurs at approximately 84 degrees C. These results correlate with the functional properties of the intramembranous section of the enzyme.

The effect of ionic strength and specific anions on substrate binding and hydrolytic activities of Na,K-ATPase.

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP3-, eosine2-, p-nitrophenylphosphate, and V(max) for Na,K-ATPase and K+-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0-0.5 M of NaCl, Na2SO4, and NaNO3 and in 0.1 M Na-acetate, NaSCN, and NaClO4. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl approximately Na2SO4 approximately Na-acetate < NaNO3 < NaSCN < NaCl04. We treated the influence of NaCl and Na2SO4 on K(diss) for E x ADP as a "pure" ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO3- effect was compatible with competitive binding of NO3- and ADP in addition to the general I-effect. K(diss) for E x NO3 was approximately 32 mM. Analysis of V(max)/K(m) for Na,K-ATPase and K+-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.

Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.

Structural integrity of the membrane domains in extensively trypsinized Na,K-ATPase from shark rectal glands.

Removal of extramembranous portions of the integral membrane protein Na,K-ATPase from shark salt glands by trypsin in the presence of Rb+ (a K+ congener) preserves the intramembranous association of the remaining membrane-spanning tryptic peptides. This is evidenced from comparison of the rotational mobility of native and trypsinized Na,K-ATPase using saturation transfer electron spin resonance spectroscopy (ESR) and from study of the lipid-protein interactions using conventional ESR spectroscopy. The interface between the lipids and the intramembranous domains is conserved on removal of the extramembranous parts of the protein, since the population of motionally restricted boundary lipids remains essentially the same in the native and trypsinized preparations. The ability to occlude Rb+ is also retained by the trypsinized membranes, as previously observed with pig kidney Na,K-ATPase. A 19-kDa fragment remaining when Na,K-ATPase is trypsinized in the presence of Rb+ is degraded further when the trypsinization is carried out in the presence of Na+ instead of Rb+. The rotational mobility of the tryptic fragments in the Na(+)-trypsinized membranes is lower than for the Rb(+)-trypsinized membranes, indicating rearrangement of the peptides. In addition, occlusion capacity is lost when trypsinization is carried out in Na+, suggesting a correlation between structure and function in the trypsinized membranes. The sequences of four membrane-spanning tryptic fragments of shark Na,K-ATPase are found to be almost identical to corresponding sequences in pig kidney Na,K-ATPase.

Influence of Na+ on conformational states in membrane-bound renal Na,K-ATPase.

Conformational states of the Na,K-ATPase and rates of transition between these are studied using the fluorescent dye eosin as a marker for the Na+ form (E1) and occlusion of 86Rb+ as a marker for the K+ form (E2). The aim of the present paper is to propose that the E1 form of the Na,K-ATPase can be liganded with a number of Rb+ and Na+ ions and that only some of these E1 forms bind eosin and thus probably nucleotides with high affinity. Experiments are performed with Na,K-ATPase isolated from pig kidney. Binding of eosin occurs only when Na+ is present at millimolar concentrations, and the observed rate of binding is slow when Rb+ is present. The rate of eosin binding after a sudden increase in the Na+ concentration is about the same as the rate of deocclusion of Rb+, suggesting that eosin monitors the rate of the E2 to E1 transition. Titrations of eosin fluorescence with Na+ indicate that binding of more than one Na+ occurs when high-affinity eosin binding takes place. With 0.05 mM RbCl and 4 mM NaCl present, the Na,K-ATPase is a mixture of at least two enzyme species which do not bind eosin with high affinity. One species is the E2 form with Rb+ occluded, and transition of this form to E1 gives rise to a small observed rate constant for eosin binding when the Na+ concentration is suddenly increased to about 25 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of poly(ethylene glycol) and aqueous viscosity on the rotational diffusion of membranous Na,K-ATPase.

The Na,K-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), E.C. 3.6.1.37] in native membranes from the salt gland of Squalus acanthias has been spin-labeled covalently with a chloromercuri nitroxide derivative, and the rotational diffusion of the protein has been studied, as a function of the concentration of glycerol or poly(ethylene glycol) in the suspending medium, by means of saturation-transfer electron spin resonance spectroscopy. The effective rotational correlation time of the protein increases linearly with the viscosity of the aqueous glycerol medium, with a gradient whose value indicates that ca. 50-70% of the volume of the Na,K-ATPase protein is external to the membrane. The effective rotational correlation times of the protein in poly(ethylene glycol) solutions are considerably greater than those in glycerol solutions of the same viscosity and increase nonlinearly with the viscosity of the suspending medium, indicating that increasing concentrations of poly(ethylene glycol) induce aggregation of the integral proteins within the membrane. The value reached at 50% poly(ethylene glycol) corresponds to a degree of aggregation of the proteins between 2 and 5 depending on whether the ethylene glycol polymer is excluded from the membrane surface region. The results are discussed with respect to hydration forces and poly(ethylene glycol)-induced cell fusion.

Maleimide, iodoacetamide, indanedione, and chloromercuric spin label reagents with derivatized nitroxide rings as ESR reporter groups for protein conformation and dynamics.

The syntheses of eight nitroxide spin labels which bear maleimide, iodoacetamide, indanedione, or chloromercuric reactive groups and, in addition, a second substituent in the nitroxide ring are presented. The second substituent groups range from hydrophobic and hydrophilic esters to carboxylic acid and secondary and tertiary amine groups. The resulting spin labels are characterized with respect both to protein covalent modification and to the electron spin resonance spectral properties of the bound labels. The effect of the various substituents in the spin label on the reactivity toward the membrane-bound shark rectal gland and pig kidney Na,K-ATPase is described. The spectral differences between immobilized and mobile groups observed by electron spin resonance for the different protein-bound spin labels show that, by selecting an appropriate derivative for modification, a large range of different motional sensitivities of the reporter group can be obtained. Such different series of spin labels should therefore be useful for detecting mobility changes arising from conformational transitions in proteins by conventional electron spin resonance spectroscopy or for measurement of protein rotational diffusion using saturation transfer electron spin resonance spectroscopy. The chloromercuric series is found to be particularly useful because of the high reactivity, the lack of reversibility that potentially is associated with the Michael addition reaction, and the wide range of rotational mobility that is exhibited by the different derivatives.