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Methods and Results in Crystallization of Membrane Proteins (Iul Biotechnology, 4)
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%content% Read the original: J. Am. Chem. Soc. In Journal of the American Chemical Society, Vol. 131, No. 35. (12 August 2009), pp. 12650-12656. We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1?36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a α-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10?100 proteins per ?m2 of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbru?ck model. At higher protein-to lipid ratios (up to 3000 ?m?2), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of ?25,000 ?m?2) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed. We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1?36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a α-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10?100 proteins per ?m2 of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbru?ck model. At higher protein-to lipid ratios (up to 3000 ?m?2), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of ?25,000 ?m?2) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed. J. Am. Chem. Soc. In Journal of the American Chemical Society, Vol. 131, No. 35. (12 August 2009), pp. 12650-12656. We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1?36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a α-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10?100 proteins per ?m2 of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbru?ck model. At higher protein-to lipid ratios (up to 3000 ?m?2), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of ?25,000 ?m?2) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed. We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1?36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a α-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10?100 proteins per ?m2 of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbru?ck model. At higher protein-to lipid ratios (up to 3000 ?m?2), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of ?25,000 ?m?2) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed. %content% Excerpt from: Mol Membr Biol In Molecular Membrane Biology, Vol. 28, No. 3. (14 February 2011), pp. 171-181. Abstract Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-?-D-maltoside (?C12-b-M?) as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b6?f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M. Abstract Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-?-D-maltoside (?C12-b-M?) as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b6?f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M. Mol Membr Biol In Molecular Membrane Biology, Vol. 28, No. 3. (14 February 2011), pp. 171-181. Abstract Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-?-D-maltoside (?C12-b-M?) as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b6?f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M. Abstract Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-?-D-maltoside (?C12-b-M?) as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b6?f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M. %content% See original here: %content% See the article here: %content% Go here to see the original: 500 Internal Server Error Excerpt from: %content% Read this article: Current medicinal chemistry, Vol. 18, No. 17. (2011), pp. 2601-2611. The binding of various molecules to integral membrane proteins with optimal affinity and specificity is central to normal function of cell. While membrane proteins represent about one third of the whole cell proteome, they are a majority of common drug targets. The quest for the development of computational models capable of accurate evaluation of binding affinities, decomposition of the binding into its principal components and thus mapping molecular mechanisms of binding remains one of the main goals of modern computational biophysics and related drug development. The primary scope of this review will be on the recent extension of computational methods for the study of drug binding to membrane proteins. Several examples of such applications will be provided ranging from secondary transporters to voltage gated channels. In this mini-review, we will provide a short summary on the breadth of different methods for binding affinity evaluation. These methods include molecular docking with docking scoring functions, molecular dynamics (MD) simulations combined with post-processing analysis using Molecular Mechanics/Poisson Boltzmann (Generalized Born) Surface Area (MM/PB(GB)SA), as well as direct evaluation of free energies from Free Energy Perturbation (FEP) with constraining schemes, and Potential of Mean Force (PMF) computations. We will compare advantages and shortcomings of popular techniques and provide discussion on the integrative strategies for drug development aimed at targeting membrane proteins. Link: %content% Follow this link: Synthetic cell membranes invented at the Institute of Materials Research and Engineering (IMRE), a research institute of Singapore’s Agency for Science, Technology and Research (A*STAR), may improve the way we identify and develop drugs by speeding up and reducing the cost of the drug screening process. The technology earned a spot as one of the twelve finalists in the Asian Innovation Awards 2011 organized by the Wall Street Journal Asia. See the original post: Proteins (2011), pp. n/a-n/a. Abstract Large-scale next generation resequencing of X chromosome genes identified a missense mutation in the CLIC2 gene on Xq28 in a male with X-linked intellectual disability (XLID) and not found in healthy individuals. At the same time, numerous nsSNPs (nonsynonomous SNP) have been reported in the CLIC2 gene in healthy individuals indicating that the CLIC2 protein can tolerate amino acid substitutions and be fully functional. To test the possibility that p.H101Q is a disease-causing mutation, we performed in silico simulations to calculate the effects of the p.H101Q mutation on CLIC2 stability, dynamics and ionization states while comparing the effects obtained for presumably harmless nsSNPs. It was found that p.H101Q, in contrast with other nsSNPs, (a) lessens the flexibility of the joint loop which is important for the normal function of CLIC2, (b) makes the overall 3D structure of CLIC2 more stable and thus reduces the possibility of the large conformational change expected to occur when CLIC2 moves from a soluble to membrane form and (c) removes the positively charged residue, H101, which may be important for the membrane association of CLIC2. The results of in silico modeling, in conjunction with the polymorphism analysis, suggest that p.H101Q may be a disease-causing mutation, the first one suggested in the CLIC family. Proteins 2011. © 2011 Wiley-Liss, Inc. |
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