Designer lipid-like peptides

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins

A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E.coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.United States. Defense Advanced Research Projects Agency (DARPA-HR0011-09-C-0012)Massachusetts Institute of Technology. Undergraduate Research Opportunities Progra

Designer Lipid-Like Peptides: A Class of Detergents for Studying Functional Olfactory Receptors Using Commercial Cell-Free Systems

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.United States. Defense Advanced Research Projects Agency (DARPA-HR0011-09-C-0012)Massachusetts Institute of Technology. Undergraduate Research Opportunities Progra

Ensuring transmission through dynamic host environments: host–pathogen interactions in <i>Plasmodium</i> sexual development

A renewed global commitment to malaria elimination lends urgency to understanding the biology of Plasmodium transmission stages. Recent progress toward uncovering the mechanisms underlying Plasmodium falciparum sexual differentiation and maturation reveals potential targets for transmission-blocking drugs and vaccines. The identification of parasite factors that alter sexual differentiation, including extracellular vesicles and a master transcriptional regulator, suggest that parasites make epigenetically controlled developmental decisions based on environmental cues. New insights into sexual development, especially host cell remodeling and sequestration in the bone marrow, highlight open questions regarding parasite homing to the tissue, transmigration across the vascular endothelium, and maturation in the parenchyma. Novel molecular and translational tools will provide further opportunities to define host–parasite interactions and design effective transmission-blocking therapeutics

Designer lipid-like peptides

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins

Silver Stains of Purified GPCRs.

<p>A) Four cell-free expressed GPCRs. B) Comparison between cell-free and HEK293 expressed hVN1R1. Most GPCRs could be purified to >90% purity, and all showed two bands characteristic of a monomer and a dimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023036#pone.0023036-Cook1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023036#pone.0023036-Cook2" target="_blank">[18]</a>. The cell-free and HEK293 expressed receptors run at the same size, and have similar purities.</p

Circular Dichroism Spectra of Five Purified GPCRs.

<p>A) Cell-free expressed mOR103-15 made with Brij-35 or no detergent, B) Cell-free expressed hTAAR5, C) Cell-free expressed hFPR3, and D) Cell-free and HEK293 expressed hVN1R1. All purified GPCRs have characteristic alphahelical spectra, except mOR103-15 made without detergent. Since GPCRs have 7-transmembrane helices, and an overall α-helix content of ∼50%, the CD spectra suggest that these receptors are properly folded. The near overlap of the spectra for cell-free and HEK293 expressed hVN1R1 suggests that both receptors are properly folded, and further indicates that cell-free produced GPCRs are comparable to those expressed in mammalian cells.</p

Solubility and maximum yields of GPCRs produced using cell-free <i>in vitro</i> translation in the presence of Brij-35.

*<p>Milligrams of receptor that could be produced in a 10 ml cell-free reaction. These yields were calculated from smaller batches of protein purified using immunoaffinity chromatography. Experiments showed that up to 1 mg/ml of protein could be produced, but that up to half could be lost during the purification process. The yields were determined by spectrophotometer readings.</p>‡<p>These yields were calculated by comparing the intensities of the receptor samples against a sample with a known concentration.</p

Detergent screen for cell-free GPCR production.

<p>Detergents commonly used for membrane protein solubilization or crystallization were screened. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023036#pone-0023036-g001" target="_blank">Figure 1</a> shows a detergent screen with the receptor hOR17-210. Brij-35 and Brij-58 yielded ∼4–5 times as much receptor as the next best detergent. Although comparable, Brij-35 consistently had slightly higher yields than Brij-58. Each bar represents the average of 2–3 experiments. The data was normalized to Brij-35.</p

Lysophosphatidylcholine regulates sexual stage differentiation in the human malaria parasite Plasmodium falciparum

Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission