Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.

Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

Comparison of the Efficiency of Ultraviolet/Ultrasonic (UV/US) and Ultraviolet/Zinc Oxide (UV/ZnO) Technologies as Advanced Oxidation Processes in the Removal of Linear Alkylbenzene Sulfonate (LAS), an Anionic Detergent, from Aqueous Media

Surfactants are one of the most commonly found xenobiotics in municipal and industrial wastewater. The purpose of this study was to compare ultraviolet/ultrasonic (UV/US) and ultraviolet/zinc oxide (UV/ZnO) processes in the removal of LAS from aqueous media.In this study, a medium-pressure UV lamp (125 W), an ultrasonic device (400 W and 42 kHz), and ZnO nanoparticles (NPs) were used. The concentration of LAS was 0.5, 14, and 21 mg/L in all experiments. Contact time of 8, 16, and 24 minutes, pH of 3, 7, and 11, and NP concentrations of 50, 100, 150 mg/L were selected. Detergent extraction was performed using methylene blue active substances.The results showed that the efficiency of UV/US process in the removal of LAS was 89.35%, while the removal efficiency of UV/ZnO process was 81.27%. In both processes, the rate of detergent removal increased by elongating the contact time from 8 to 24 minutes. The efficiency of UV/US process in LAS removal was greater than that of the UV/ZnO process. The findings showed that the removal efficiency of UV/US process was directly correlated with pH, while it had an inverse correlation with the removal efficiency of UV/ZnO process.

Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Membrane proteins are essential for cellular growth and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates significant challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a significant advantage. Here we use a novel type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful detergent-free approach for the study of membrane proteins allowing native extraction and high-resolution structure determination by cryoEM.

Evidence for Membrane Complex Assembly in Nanoelectrospray Generated Lipid Bilayers

1.AbstractNanoelectrospray can be used to generate a layered structure consisting of bipolar lipids, detergent-solubilized membrane proteins, and glycerol that self-assembles upon detergent extraction into one extended layer of a protein containing membrane. This manuscript presents the first evidence that this method might allow membrane protein complexes to assemble in this process.

Formulation of Paint and Detergent with Pelargonium citrosum & Rosmarinus officinalis Extracts as Musca domestica Repellent Agent

Use of botanical environmentally friendly and biodegradable insect repellants as opposed to chemical insecticides is increasingly becoming important as an alternative method of insect control. Housefly (Musca domestica L.) has potential of transmitting pathogen causing diseases such as typhoid, cholera, bacillary dysentery, tuberculosis, anthrax, ophthalmia, and parasitic worms. Essential oils derived from aromatic plants have exhibited biological properties and can be used to prevent and treat human diseases. The aim of this study was to formulate the housefly repellent paint and detergent. Extraction of essential oils was by hydro-distillation. Condensed oil extracts were collected in n-hexane and insect behavioral response tested using adult houseflies (Musca domestica L.). N, N-diethyl-m-toluamide (DEET) was used as the positive control and acetone as the negative control. The formulated products showed higher repellent activities against the housefly. When 1% (V/V %) insect repellent detergent and paint were compared, the housefly repellent activities occurred at 67±2.64 for detergent and 76±1.34 for the paint. When 3% (V/V%) insect repellent detergent and paint were compared, the housefly repellent activities occurred at 81±1.77 for detergent and 84±2.1 for the paint for outdoor and indoor field trials which were carried out for eight hours each day for two days. The results provide scientific rationale for traditional use of R. officinalis oil and Pelargonium citrosum in control of housefly and other common insects in the household.