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Michael R. Jones - One of the best experts on this subject based on the ideXlab platform.
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the effectiveness of styrene maleic acid sma copolymers for solubilisation of Integral Membrane Proteins from sma accessible and sma resistant Membranes
Biochimica et Biophysica Acta, 2017Co-Authors: David J K Swainsbury, Nicholas Foster, Rienk Van Grondelle, S Scheidelaar, Antoinette J Killian, Michael R. JonesAbstract:Abstract Solubilisation of biological lipid bilayer Membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of Integral Membrane Proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic Membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (
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The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of Integral Membrane Proteins from SMA-accessible and SMA-resistant Membranes
Biochimica et Biophysica Acta - Biomembranes, 2017Co-Authors: David J K Swainsbury, Nicholas Foster, Rienk Van Grondelle, S Scheidelaar, J. Antoinette Killian, Michael R. JonesAbstract:Solubilisation of biological lipid bilayer Membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of Integral Membrane Proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic Membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (< 30 kDa weight average molecular weight). The effectiveness of 10 kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic Membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, Proteins isolated from such Membranes comprised clusters of complexes in small Membrane patches rather than individual Proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising Integral Membrane Proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of Proteins in the Membrane.
David J K Swainsbury - One of the best experts on this subject based on the ideXlab platform.
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the effectiveness of styrene maleic acid sma copolymers for solubilisation of Integral Membrane Proteins from sma accessible and sma resistant Membranes
Biochimica et Biophysica Acta, 2017Co-Authors: David J K Swainsbury, Nicholas Foster, Rienk Van Grondelle, S Scheidelaar, Antoinette J Killian, Michael R. JonesAbstract:Abstract Solubilisation of biological lipid bilayer Membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of Integral Membrane Proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic Membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (
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The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of Integral Membrane Proteins from SMA-accessible and SMA-resistant Membranes
Biochimica et Biophysica Acta - Biomembranes, 2017Co-Authors: David J K Swainsbury, Nicholas Foster, Rienk Van Grondelle, S Scheidelaar, J. Antoinette Killian, Michael R. JonesAbstract:Solubilisation of biological lipid bilayer Membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of Integral Membrane Proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic Membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (< 30 kDa weight average molecular weight). The effectiveness of 10 kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic Membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, Proteins isolated from such Membranes comprised clusters of complexes in small Membrane patches rather than individual Proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising Integral Membrane Proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of Proteins in the Membrane.
Julian P. Whitelegge - One of the best experts on this subject based on the ideXlab platform.
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Integral Membrane Proteins bottom up top down and structural proteomics
Expert Review of Proteomics, 2017Co-Authors: Upendra K Kar, Margaret Simonian, Julian P. WhiteleggeAbstract:AbstractIntroduction: Integral Membrane Proteins and lipids constitute the bilayer Membranes that surround cells and sub-cellular compartments, and modulate movements of molecules and information between them. Since Membrane protein drug targets represent a disproportionately large segment of the proteome, technical developments need timely review.Areas covered and literature search strategy: Publically available resources such as Pubmed were surveyed. Bottom-up proteomics analyses now allow efficient extraction and digestion such that Membrane protein coverage is essentially complete, making up around one third of the proteome. However, this coverage relies upon hydrophilic loop regions while transMembrane domains are generally poorly covered in peptide-based strategies. Top-down mass spectrometry where the intact Membrane protein is fragmented in the gas phase gives good coverage in transMembrane regions, and Membrane fractions are yielding to high-throughput top-down proteomics. Exciting progress in na...
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Integral Membrane Proteins and Bilayer Proteomics
Analytical chemistry, 2013Co-Authors: Julian P. WhiteleggeAbstract:Integral Membrane Proteins reside within the bilayer Membranes that surround cells and organelles, playing critical roles in movement of molecules across them and the transduction of energy and sig...
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Integral Membrane Proteins and bilayer proteomics
Analytical Chemistry, 2013Co-Authors: Julian P. WhiteleggeAbstract:Integral Membrane Proteins reside within the bilayer Membranes that surround cells and organelles, playing critical roles in movement of molecules across them and the transduction of energy and signals. While their extreme amphipathicity presents technical challenges, biological mass spectrometry has been applied to all aspects of Membrane protein chemistry and biology, including analysis of primary, secondary, tertiary and quaternary structure, as well as the dynamics that accompany functional cycles and catalysis.
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profiling of Integral Membrane Proteins and their post translational modifications using high resolution mass spectrometry
Methods, 2011Co-Authors: Puneet Souda, Christopher M. Ryan, Julian P. WhiteleggeAbstract:Integral Membrane Proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transMembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transMembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a Membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transMembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic Integral Membrane Proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transMembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation.
Nicholas Foster - One of the best experts on this subject based on the ideXlab platform.
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the effectiveness of styrene maleic acid sma copolymers for solubilisation of Integral Membrane Proteins from sma accessible and sma resistant Membranes
Biochimica et Biophysica Acta, 2017Co-Authors: David J K Swainsbury, Nicholas Foster, Rienk Van Grondelle, S Scheidelaar, Antoinette J Killian, Michael R. JonesAbstract:Abstract Solubilisation of biological lipid bilayer Membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of Integral Membrane Proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic Membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (
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The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of Integral Membrane Proteins from SMA-accessible and SMA-resistant Membranes
Biochimica et Biophysica Acta - Biomembranes, 2017Co-Authors: David J K Swainsbury, Nicholas Foster, Rienk Van Grondelle, S Scheidelaar, J. Antoinette Killian, Michael R. JonesAbstract:Solubilisation of biological lipid bilayer Membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of Integral Membrane Proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic Membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (< 30 kDa weight average molecular weight). The effectiveness of 10 kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic Membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, Proteins isolated from such Membranes comprised clusters of complexes in small Membrane patches rather than individual Proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising Integral Membrane Proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of Proteins in the Membrane.
Rienk Van Grondelle - One of the best experts on this subject based on the ideXlab platform.
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the effectiveness of styrene maleic acid sma copolymers for solubilisation of Integral Membrane Proteins from sma accessible and sma resistant Membranes
Biochimica et Biophysica Acta, 2017Co-Authors: David J K Swainsbury, Nicholas Foster, Rienk Van Grondelle, S Scheidelaar, Antoinette J Killian, Michael R. JonesAbstract:Abstract Solubilisation of biological lipid bilayer Membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of Integral Membrane Proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic Membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (
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The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of Integral Membrane Proteins from SMA-accessible and SMA-resistant Membranes
Biochimica et Biophysica Acta - Biomembranes, 2017Co-Authors: David J K Swainsbury, Nicholas Foster, Rienk Van Grondelle, S Scheidelaar, J. Antoinette Killian, Michael R. JonesAbstract:Solubilisation of biological lipid bilayer Membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of Integral Membrane Proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic Membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (< 30 kDa weight average molecular weight). The effectiveness of 10 kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic Membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, Proteins isolated from such Membranes comprised clusters of complexes in small Membrane patches rather than individual Proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising Integral Membrane Proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of Proteins in the Membrane.