1,534 research outputs found
Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.
No full textPathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli
Editorial: Verification and Validation of in silico Models for Biomedical Implantable Devices
No full textAtomic force microscopy for the study of membrane proteins
No full textFundamental biological processes such as cell-cell communication, signal transduction, molecular transport and energy conversion are performed by membrane proteins. These important proteins are studied best in their native environment, the lipid bilayer. The atomic force microscope (AFM) is the instrument of choice to determine the native surface structure, supramolecular organization, conformational changes and dynamics of membrane-embedded proteins under near-physiological conditions. In addition, membrane proteins are imaged at subnanometer resolution and at the single molecule level with the AFM. This review highlights the major advances and results achieved on reconstituted membrane proteins and native membranes as well as the recent developments of the AFM for imaging
Two-Dimensional Crystallisation of Membrane Proteins and Structural Assessment
No full textTwo-dimensional (2D) crystallisation of Membrane proteins reconstitutes them into their native environment, the lipid bilayer. Electron crystallography
allows the structural analysis of these regular protein–lipid arrays up to atomic resolution. The crystal quality depends on the protein purity, ist stability and on the crystallisation conditions. The basics of 2D crystallisation and different recent advances are reviewed and electron crystallography approaches summarised. Progress in 2D crystallisation, sample preparation, image detectors and automation of the data acquisition and processing pipeline makes 2D electron crystallography particularly attractive for the structural analysis
of membrane proteins that are too small for single-particle analyses and too unstable to form three-dimensional (3D) crystals
Mining motor symptoms UPDRS data of Parkinson's disease patients for the development of Hoehn and Yahr estimation decision support system
No full text
Secondary Active Transporters.
No full textTransport of solutes across biological membranes is essential for cellular life. This process is mediated by membrane transport proteins which move nutrients, waste products, certain drugs and ions into and out of cells. Secondary active transporters couple the transport of substrates against their concentration gradients with the transport of other solutes down their concentration gradients. The alternating access model of membrane transporters and the coupling mechanism of secondary active transporters are introduced in this book chapter. Structural studies have identified typical protein folds for transporters that we exemplify by the major facilitator superfamily (MFS) and LeuT folds. Finally, substrate binding and substrate translocation of the transporters LacY of the MFS and AdiC of the amino acid-polyamine-organocation (APC) superfamily are described
Preparation of detergent-solubilized membranes from Escherichia coli
No full textAuthors: Daniel Harder & Dimitrios Fotiadis
### Abstract
This protocol describes a method to prepare detergent-solubilized membranes from Escherichia coli (E. coli), e.g. containing an overexpressed membrane protein. The procedure takes less than one day. Cells are broken by pressure cell and membranes are isolated and washed by differential centrifugation. Finally, the membranes are solubilized with the detergent of choice.
### Reagents
1. Cell pellet of E. coli
- Tris
- EDTA
- NaCl
- n-dodecyl-β-D-maltoside (DDM) or another detergent of choice
glycerol
- NaN3
### Equipment
1. French Press
- ultracentrifuge
### Procedure
1. Resuspend the cell pellet from 1 l of E. coli culture in Lysis Buffer (20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA). Adapt the buffer volume to your French pressure cell.
- Disrupt E. coli cells by passage through a French pressure cell (20,000 psi) and remove unbroken cells by centrifugation at 10,000g (4˚C, 10 min).
- Ultracentrifuge the supernatant at 100,000g (4˚C, 1 h).
- Resuspend and homogenize the pellet containing the E. coli membranes in Lysis Buffer and ultracentrifuge again.
- Remove water-soluble proteins adhering to the membrane by homogenization in 20 mM Tris-HCl, pH 8.0, 300 mM NaCl and ultracentrifugation.
- Breakpoint: Membrane pellet can be resuspended in 1 ml buffer without detergent and stored at -80 °C for months to years prior detergent solubilization.
- Resuspend and solubilize the membrane pellet in 1% DDM, 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 0.01% NaN3 for 2 h at 4˚C under gentle agitation (final volume: 7 ml).
- Ultracentrifuge at 100,000g (4˚C, 50 min).
- The supernatant represents solubilized membranes, which can be used for purification of the His-tagged protein or SPA-binding experiments directly.
### Timing
The procedure takes less than one day.
### References
- Casagrande, F. et al. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily. *J. Biol. Chem*. 283, 33240-33248 (2008).
### Associated Publications
1. **Projection Structure of a Member of the Amino Acid/Polyamine/Organocation Transporter Superfamily**. F. Casagrande, M. Ratera, A. D. Schenk, M. Chami, E. Valencia, J. M. Lopez, D. Torrents, A. Engel, M. Palacin, and D. Fotiadis. *Journal of Biological Chemistry* 283 (48) 33240 - 33248 23/09/2008 doi:10.1074/jbc.M806917200
- **Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay**. Daniel Harder and Dimitrios Fotiadis. *Nature Protocols* 7 (9) 1569 - 1578 doi:10.1038/nprot.2012.090
### Author information
**Daniel Harder & Dimitrios Fotiadis**, Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland
Correspondence to: Dimitrios Fotiadis ([email protected])
*Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2395) (2012) doi:10.1038/protex.2012.033. Originally published online 7 August 2012*
High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.
No full textAtomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures
Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2.
No full textHeterodimeric amino acid transporters (HATs) are protein complexes mediating the transport of amino acids and derivatives thereof across biological membranes. HATs are composed of two subunits, a heavy and a light chain subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. The human HAT 4F2hc-LAT2 is composed of the type-II membrane N-glycoprotein 4F2hc (SCL3A2) and the L-type amino acid transporter LAT2 (SLC7A8), which are covalently linked to each other by a conserved disulfide bridge. Whereas LAT2 catalyzes substrate transport, 4F2hc is important for the successful trafficking of the transporter to the plasma membrane. The overexpression, malfunction, or absence of 4F2hc-LAT2 is associated with human diseases, and therefore, this heterodimeric complex represents a potential drug target. The recombinant human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris, and the protein can be purified. Here, we present the cryo-EM density map of the human 4F2hc-LAT2 amino acid transporter at sub-nanometer resolution. A homology model of 4F2hc-LAT2 in the inward-open conformation was generated and fitted into the cryo-EM density and analyzed. In addition, disease-causing point mutations in human LAT2 were mapped on the homology model of 4F2hc-LAT2, and the possible functional implications on the molecular level are discussed
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