Conformational Analysis of Membrane Proteins in Phospholipid Bilayer Nanodiscs by Hydrogen Exchange Mass Spectrometry

The study of membrane protein structure and enzymology has traditionally been hampered by the inherent insolubility of membrane proteins in aqueous environments and experimental challenges in emulating an in vivo lipid environment. Phospholipid bilayer nanodiscs have recently been shown to be of great use for the study of membrane proteins since they offer a controllable, stable, and monodisperse model membrane with a native-like lipid bilayer. Here we report the integration of nanodiscs with hydrogen exchange (HX) mass spectrometry (MS) experiments, thereby allowing for analysis of the native conformation of membrane proteins. Gamma-glutamyl carboxylase (GGCX), an ~94 kDa transmembrane protein, was inserted into nanodiscs and labeled with deuterium oxide under native conditions. Analytical parameters including sample-handling and chromatographic separation were optimized to measure the incorporation of deuterium into GGCX. Coupling nanodisc technology with HX MS offers an effective approach for investigating the conformation and dynamics of membrane proteins in their native environment and is therefore capable of providing much needed insight into the function of membrane proteins

Conformational Transitions in the Membrane Scaffold Protein of Phospholipid Bilayer Nanodiscs

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest

A Long Slit-Like Entrance Promotes Ventilation in the Mud Nesting Social Wasp, Polybia spinifex: Visualization of Nest Microclimates using Computational Fluid Dynamics

Polybia spinifex Richards (Hymenoptera: Vespidae) constructs mud nests characterized by a long slit-like entrance. The ventilation and thermal characteristics of the P. spinifex nest were investigated to determine whether the nest microclimate is automatically maintained due to the size of the entrance. In order to examine this hypothesis, a numerical simulation was employed to predict the effects of the entrance length. The calculations were performed with 3D-virtual models that simulated the P. spinifex nest conditions, and the reliability of the simulations was experimentally examined by using gypsum-model nests and a P. spinifex nest. The ventilation effect was determined by blowing air through the nest at 1–3 m/s (airflow conditions); the airspeed was found to be higher in models with a longer entrance. The ventilation rate was also higher in models with longer entrances, suggesting that the P. spinifex nest is automatically ventilated by natural winds. Next, the thermal effect was calculated under condition of direct sunlight. Under a calm condition (airflow, 0 m/s), thermal convection and a small temperature drop were observed in the case of models with a long entrance, whereas the ventilation and thermoregulation effects seemed small. Under airflow conditions, the temperature at the mid combs steeply dropped due to the convective airflow through the entrance at 1–2 m/s, and at 3 m/s, most of the heat was eliminated due to high thermal conductivity of the mud envelope, rather than convection

Metamaterial-Enhanced Nonlinear Terahertz Spectroscopy

We demonstrate large nonlinear terahertz responses in the gaps of metamaterial split ring resonators in several materials and use nonlinear THz transmission and THz-pump/THz-probe spectroscopy to study the nonlinear responses and dynamics. We use the field enhancement in the SRR gaps to initiate high-field phenomena at lower incident fields. In vanadium dioxide, we drive the insulator-to-metal phase transition with high-field THz radiation. The film conductivity increases by over two orders of magnitude and the phase transition occurs on a several picosecond timescale. In gallium arsenide, we observe high-field transport phenomena, including mobility saturation and impact ionization. The carrier density increases by up to ten orders of magnitude at high fields. At the highest fields, we demonstrate THz-induced damage in both vanadium dioxide and gallium arsenide.United States. Dept. of Energy (DOE-BES, grant DE-FG02- 09ER46643)United States. Office of Naval Research (ONR Grant No. N00014-09-1-1103

Cherenkov radiation emitted by ultrafast laser pulses and the generation of coherent polaritons

We report on the generation of coherent phonon polaritons in ZnTe, GaP and LiTaO$_{3}$ using ultrafast optical pulses. These polaritons are coupled modes consisting of mostly far-infrared radiation and a small phonon component, which are excited through nonlinear optical processes involving the Raman and the second-order susceptibilities (difference frequency generation). We probe their associated hybrid vibrational-electric field, in the THz range, by electro-optic sampling methods. The measured field patterns agree very well with calculations for the field due to a distribution of dipoles that follows the shape and moves with the group velocity of the optical pulses. For a tightly focused pulse, the pattern is identical to that of classical Cherenkov radiation by a moving dipole. Results for other shapes and, in particular, for the planar and transient-grating geometries, are accounted for by a convolution of the Cherenkov field due to a point dipole with the function describing the slowly-varying intensity of the pulse. Hence, polariton fields resulting from pulses of arbitrary shape can be described quantitatively in terms of expressions for the Cherenkov radiation emitted by an extended source. Using the Cherenkov approach, we recover the phase-matching conditions that lead to the selection of specific polariton wavevectors in the planar and transient grating geometry as well as the Cherenkov angle itself. The formalism can be easily extended to media exhibiting dispersion in the THz range. Calculations and experimental data for point-like and planar sources reveal significant differences between the so-called superluminal and subluminal cases where the group velocity of the optical pulses is, respectively, above and below the highest phase velocity in the infrared.Comment: 13 pages, 11 figure

A New Peptide Ligand for Targeting Human Carbonic Anhydrase IX, Identified through the Phage Display Technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy