Probing the U-Shaped Conformation of Caveolin-1 in a Bilayer

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82–S136; Cav1_82–136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav1_82–136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav1_82–136 insertion depth in the bilayer), corroborate that Cav1_82–136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav1_82–136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.     

A Method for Cell Culture and Maintenance of Ammonia-Oxidizing Archaea in Agar Stab

Ammonia oxidizing archaea (AOA) are predominantly found and closely linked with geochemical cycling of nitrogen in non-extreme habitats. However, these strains have mainly been investigated using liquid cultures of enriched cells. Here, we provide an agar stab as a simple and reliable means of cultivating and maintaining AOA.     

Surface Chirality of Gly‐Pro Dipeptide Adsorbed on a Cu(110) Surface

The adsorption of chiral Gly‐Pro dipeptide on Cu(110) has been characterized by combining in situ polarization modulation infrared reflection absorption spectroscopy (PM‐RAIRS) and X‐ray photoelectron spectroscopy (XPS). The chemical state of the dipeptide, and its anchoring points and adsorption geometry, were determined at various coverage values. Gly‐Pro molecules are present on Cu(110) in their anionic form (NH₂/COO⁻) and adsorb under a 3‐point binding via both oxygen atoms of the carboxylate group and via the nitrogen atom of the amine group. Low‐energy electron diffraction (LEED) and scanning tunneling microscopy (STM) have shown the presence of an extended 2D chiral array, sustained via intermolecular H‐bonds interactions. Furthermore, due to the particular shape of the molecule, only one homochiral domain is formed, creating thus a truly chiral surface. Chirality 27:411–416, 2015. © 2015 Wiley Periodicals, Inc.     

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.     

Lipid nanotechnologies for structural studies of membrane‐associated proteins

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane‐associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo‐electron microscopy (cryo‐EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ～20 nm inner diameter and a few microns in length, that self‐assemble in aqueous solutions. The lipid nanodisks (NDs) are self‐assembled discoid lipid bilayers of ～10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane‐associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane‐bound coagulation factor VIII in vitro for structure determination by cryo‐EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three‐dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane‐associated proteins and complexes for structural studies by cryo‐EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane‐associated proteins, such as the coagulation factors, at a close to physiological environment. Proteins 2014; 82:2902–2909. © 2014 Wiley Periodicals, Inc.     

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.