Papers by Simon G Patching
International Journal of Current Science Research and Review, Jan 20, 2025
Nuclear magnetic resonance (NMR) spectroscopy is a powerful non-invasive analytical technique wit... more Nuclear magnetic resonance (NMR) spectroscopy is a powerful non-invasive analytical technique with wide applications that can observe multiple nuclear species at a site-resolved level. Despite this, NMR has inherent low sensitivity compared to other analytical techniques. A principal approach to improve the sensitivity and resolution of the NMR experiment is to increase the strength of the external static magnetic field (B0), for which the upper practicable limit has gradually increased over five decades. The relatively recent use of high-temperature superconducting materials, such as Bi2Sr2Ca2Cu3Ox (Bi-2223), Bi2Sr2CaCu2Ox (Bi-2212) or REBa2Cu3O7-x (REBCO, RE = rare earth), has enabled construction of ultra-high field NMR magnets. Over twenty commercial ultra-high field NMR instruments at 1.0, 1.1 and 1.2 GHz (23.5, 25.9 and 28.2 Tesla, respectively) have been installed worldwide in the past several years, with more to come. NMR at ultra-high fields benefits both solution-state and solid-state NMR applications. The potential improvements in sensitivity and resolution in NMR spectra are particularly important for studying the structure, dynamics and ligand interactions of biomolecules, which can suffer from poor sensitivity and prohibitive signal crowding. The benefits of using ultra-high field NMR have begun to be demonstrated on various sample types, including intrinsically disordered proteins, membrane proteins, amyloid fibrils, viral capsids, bacterial chlorosomes, fungal cell walls, and whole human cells. Alongside optimisations in sample preparation, probe design and pulse sequences, and exploitation of dynamic nuclear polarisation (DNP), ultra-high field magnets are contributing to an exciting period for improving the sensitivity and resolution of NMR spectra in the study of more complex biomolecules and other samples.
Journal of Biological Research - Bollettino della Società Italiana di Biologia Sperimentale, 2025
Tribulus terrestris is a member of the family Zygophyllaceae commonly known as “puncture vine”. T... more Tribulus terrestris is a member of the family Zygophyllaceae commonly known as “puncture vine”. The plant has been used traditionally as an analgesic and to relieve rheumatic pain, eye problems, sexual dysfunction and edema. The aim of this work was to test the use of T. terrestris fruit extract as a reducing agent in synthesizing gold nanoparticles (AuNPs), test their biological activities, and assess their suitability as a therapeutic agent by testing them for potential adverse effects on human cells. Indeed, we have performed the most comprehensive biological testing of AuNPs produced using T. terrestris extracts to date. The aqueous extract of dried powdered T. terrestris fruits was used for the reduction of hydrogen tetrachloroaurate (III) trihydrate (AuCl4·3H2O). The fruit extract's phytochemical components effectively served as reducing, capping and stabilizing agents, resulting in the production of consistent and round-shaped AuNPs with a size range of less than 100 nm. The synthesized AuNPs were subjected to various physicochemical analyses, then evaluated for antibacterial, antifungal and antileishmanial activity, and subjected to hemagglutination, cytotoxicity and antioxidant bioassays. The AuNPs showed inhibition zones against several bacterial and fungal strains, and exhibited antileishmanial activity at high doses. The AuNPs demonstrated positive hemagglutination activity against human Red Blood Cells (RBCs) of blood groups A and B at 10 and 20 µg/mL, but no hemagglutination activity against groups AB and O at up to 40 µg/mL. The AuNPs showed no cytotoxicity against human RBCs at up to 40 µg/mL, suggesting that they may be suitable for use in a clinical setting. The antioxidant activity of the AuNPs was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and the results indicated a high antioxidant potential.
CSD Communication, 2012
Crystal structure of 5-(1H-indol-3-ylmethyl)imidazolidine-2,4-dione monohydrate.
An entry from... more Crystal structure of 5-(1H-indol-3-ylmethyl)imidazolidine-2,4-dione monohydrate.
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
OSF Preprints, 2024
Nuclear magnetic resonance (NMR) spectroscopy is a powerful non-invasive analytical technique wit... more Nuclear magnetic resonance (NMR) spectroscopy is a powerful non-invasive analytical technique with wide applications that can observe multiple nuclear species at a site-resolved level. Despite this, NMR has inherent low sensitivity compared to other analytical techniques. A principal approach to improve the sensitivity and resolution of the NMR experiment is to increase the strength of the external static magnetic field (B0), for which the upper practicable limit has gradually increased over five decades. The relatively recent use of hightemperature superconducting materials, such as Bi2Sr2Ca2Cu3Ox (Bi-2223), Bi2Sr2CaCu2Ox (Bi-2212) or REBa2Cu3O7-x (REBCO, RE = rare earth), has enabled construction of ultra-high field NMR magnets. Over twenty commercial ultra-high field NMR instruments at 1.0, 1.1 and 1.2 GHz (23.5, 25.9 and 28.2 Tesla, respectively) have been installed worldwide in the past several years, with more to come. NMR at ultra-high fields benefits both solution-state and solid-state NMR applications. The potential improvements in sensitivity and resolution in NMR spectra are particularly important for studying the structure, dynamics and ligand interactions of biomolecules, which can suffer from poor sensitivity and prohibitive signal crowding. The benefits of using ultra-high field NMR have begun to be demonstrated on various sample types, including intrinsically disordered proteins, membrane proteins, amyloid fibrils, viral capsids, bacterial chlorosomes, fungal cell walls, and whole human cells. Alongside optimisations in sample preparation, probe design and pulse sequences, and exploitation of dynamic nuclear polarisation (DNP), ultra-high field magnets are contributing to an exciting period for improving the sensitivity and resolution of NMR spectra in the study of more complex biomolecules and other samples.
![Research paper thumbnail of Roles of Nucleoside Transporters and the Nucleoside Analogue Radiotracer 3'-Deoxy-3'-[18F]Fluoro-L-Thymidine ([18F]FLT) in PET Imaging of Cancer Cells](https://attachments.academia-assets.com/118474993/thumbnails/1.jpg)
Medicine and Medical Research: New Perspectives, 2024
The present study highlights nucleoside transporters in PET imaging of proliferating cancer cells... more The present study highlights nucleoside transporters in PET imaging of proliferating cancer cells using 3'-deoxy-3'-[18F]fluoro-L-thymidine ([18F]FLT). Nucleoside transport has an important role in maintaining the hyperproliferative state of most tumors and is therefore an important target for diagnostic and therapeutic agents in the detection, treatment and monitoring of cancers. Nucleoside transport proteins facilitate the transfer of physiological nucleosides and nucleoside analogue medications across biological membranes. Nucleoside transporters are key targets for therapeutic and diagnostic drugs used in the identification, management, and surveillance of cancers. They play a significant role in the maintenance of the hyperproliferative state of tumors in cancer. The nucleoside-based probe ([18F]FLT) has been developed for PET imaging of proliferating cancer cells, which is less prone than 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) to non-specific effects. [18F]FLT enters proliferating cells through nucleoside transporters, then becomes phosphorylated and blocks DNA synthesis, whilst also becoming trapped inside the cell. Practicable and automated chemical syntheses of [18F]FLT have been developed, for which the most widely used radiolabelling precursor is the thymidine derivative 3-N-boc-5'-O-dimethoxytrityl-3'-O-nosyl-thymidine. Competitive cellular inhibition experiments with [18F]FLT suggested an involvement of hENT2 (in addition to hENT1) in the uptake of [18F]FLT in breast cancer cells, and also an involvement of hCNT1 and hCNT2. Further studies are required to investigate the potential role of hENT2 as a biomarker for breast cancer and to identify hENT2-specific inhibitors in targeted breast cancer therapy. [18F]FLT PET imaging has undergone feasibility studies and has been assessed in pre-clinical and clinical studies for the detection and diagnosis of various types of cancers and in monitoring their response to treatments. The roles of nucleoside transporters, especially ENT1, in the cellular uptake of [18F]FLT have been investigated.
Chemical Science International Journal, 2024
Microscale thermophoresis (MST) is an analytical technique for measuring biomolecular interaction... more Microscale thermophoresis (MST) is an analytical technique for measuring biomolecular interactions. It is based on the physical phenomenon that particles move within temperature gradients, which is affected by their size, charge, hydration shell and conformation. The MST sample must contain a fluorescent target molecule used to observe the movement of particles, and this can be titrated with an unlabelled binding partner for quantifying the interaction. MST is highly sensitive, using relatively small amounts of sample, and it has no limitations on the size of the target biomolecule, on the affinity of the interaction or on the composition of the buffer and other sample components. This makes MST ideally suited to characterising interactions with membrane proteins, which can be studied in cell lysates, native membranes, solubilised in detergents or reconstituted in lipids. The intrinsic aromatic residues of membrane proteins have been used as the fluorophore for MST (label-free MST) or membrane proteins have been labelled with a range of fluorescent dyes or conjugated with fluorescent proteins (labelled MST). The different types of membrane proteins that have had biomolecular interactions characterised by MST include the SARS-CoV-2 spike protein, GPCRs and other receptors, sensor kinases, ion channels, aquaporins, and transport proteins.
International Journal of Current Science Research and Review , Jun 3, 2024
The nucleoside transporter NupG is one of the two principal transport proteins in the inner membr... more The nucleoside transporter NupG is one of the two principal transport proteins in the inner membrane of Escherichia coli that enable the organism to scavenge nucleosides from its external environment. NupG functions in a symport manner driven by the proton motive force and is a member of the Nucleoside:H+ Symporter (NHS) subfamily of the Major Facilitator Superfamily (MFS) of transporters. NupG has broad substrate specificity, transporting all naturally occurring purine and pyrimidine nucleosides. In studies over many years the nupG gene has been cloned and amplified, and the NupG protein has been purified, subjected to biochemical, biophysical and computational analysis, and its X-ray structure determined in the apo state at 3.0 Å resolution. The NupG structure had a typical MFS fold with twelve transmembrane spanning α-helices and distinct N- and C-terminal domains linked by a flexible loop. Preliminary site-directed mutagenesis and molecular docking studies on NupG identified nine putative nucleoside binding pocket residues (R136, T140, F143, Q225, N228, Q261, E264, Y318, F322) and a mutant (D323A) with 20-fold enhanced uridine binding activity. Further biochemical and structural investigations are necessary to better understand the substrate recognition and molecular mechanism of E. coli NHS family proteins (NupG, XapB, YegT).
viXra Preprints, 2024
Multidrug efflux proteins, also known as efflux pumps, are one of the major mechanisms that bacte... more Multidrug efflux proteins, also known as efflux pumps, are one of the major mechanisms that bacteria have evolved for their resistance against antimicrobial agents. Gram-negative bacteria are intrinsically more resistant to many antibiotics and biocides due to their cell structure and the activity of multidrug efflux proteins. These transporters actively extrude antibiotics and other xenobiotics from the cytoplasm or surrounding membranes of cells to the external environment. Based on amino acid sequence similarity, substrate specificity and the energy source used to export their substrates, there are seven major families of distinct bacterial multidrug efflux proteins: ABC, RND, MFS, SMR, MATE, PACE, AbgT. Individual proteins may be highly specialized for one compound or highly promiscuous, transporting a broad range of structurally dissimilar substrates. Protein structural organization in a large majority of the families, including the number of transmembrane helices, has been confirmed by high-resolution structure determination for at least one member. In this book chapter, we provide an updated review on the families of bacterial multidrug efflux proteins, including basic properties, energization, structural organization and molecular mechanism. Using representative proteins from each family, we also performed analyses of transmembrane helices, amino acid composition and distribution of charged residues. Ongoing characterization of structure-function relationships and regulation of bacterial multidrug efflux proteins are necessary for contributing new knowledge to assist drug development and strategies that will overcome antimicrobial resistance.
OSF Preprints, 2024
The nucleoside transporter NupG is one of the two principal transport proteins in the inner membr... more The nucleoside transporter NupG is one of the two principal transport proteins in the inner membrane of Escherichia coli that enable the organism to scavenge nucleosides from its external environment. NupG functions in a symport manner driven by the proton motive force and is a member of the Nucleoside:H+ Symporter (NHS) subfamily of the Major Facilitator Superfamily (MFS) of transporters. NupG has broad substrate specificity, transporting all naturally occurring purine and pyrimidine nucleosides. In studies over many years the nupG gene has been cloned and amplified, and the NupG protein has been purified, subjected to biochemical, biophysical and computational analysis, and its X-ray structure determined in the apo state at 3.0 Å resolution. The NupG structure had a typical MFS fold with twelve transmembrane spanning α-helices and distinct N-and C-terminal domains linked by a flexible loop. Preliminary site-directed mutagenesis and molecular docking studies on NupG identified nine putative nucleoside binding pocket residues (R136, T140, F143, Q225, N228, Q261, E264, Y318, F322) and a mutant (D323A) with 20-fold enhanced uridine binding activity. Further biochemical and structural investigations are necessary to better understand the substrate recognition and molecular mechanism of E. coli NHS family proteins (NupG, XapB, YegT).
Innovations in Biological Science, 2024
The present study primarily focuses on "Polarising Agents and Spin Tags for Dynamic Nuclear Polar... more The present study primarily focuses on "Polarising Agents and Spin Tags for Dynamic Nuclear Polarisation (DNP)-Enhanced Solid-State Nuclear Magnetic Resonance (ssNMR) Analysis of Biological Samples". For biological materials that are frozen or have a solid-like consistency, ssNMR spectroscopy can provide structural, functional, and ligand-binding information. ssNMR spectra can be greatly improved with the application of dynamic nuclear polarisation (DNP). Through microwave irradiation at or near the electron Larmor frequency, polarisation transfer from high-gyromagnetic ratio () unpaired electrons to neighboring nuclei occurs in DNP. This produces an absolute increase in the signal-to-noise ratio and allows experiments on much smaller quantities of sample and/or using much shorter acquisition times. Along with necessary instrumentation an essential requirement for DNP-ssNMR is a sample with an endogenous free radical or an exogenous free radical polarising agent must be added to the sample. The polarising agent must be soluble in the sample matrix and compatible with the biological sample. The free radical(s) of the polarising agent also must be stable for the lifetime of DNP-ssNMR experiments. Nitroxides have been most used as polarising agents, including the biradical compounds TOTAPOL and AMUPol with a wide range of biological samples to produce DNP enhancement factors () of up to 250. Derivatives of TOTAPOL and AMUPol and many other different polarising agents have also been used. Whilst conventional polarising agents are mixed throughout the sample, others are targeted at specific sites to provide a more localised signal enhancement. Here we review the different polarising agents and spin tags that have been used in DNP-ssNMR studies on biological samples. Targeted polarising agents enable use of matrix-free samples to concentrate the sample, whilst others can be covalently bound to provide signal enhancement at highly specific sites. The continued development of novel polarising agents and labelling and sample preparation strategies for DNP-ssNMR can open many biological samples to NMR studies that were not previously possible.
Journal of the American Chemical Society, Feb 21, 2004
We have devised methods in which cross-polarization magic-angle spinning (CP-MAS) solidstate NMR ... more We have devised methods in which cross-polarization magic-angle spinning (CP-MAS) solidstate NMR is exploited to measure rigorous parameters for binding of 13 C-labeled substrates to membrane transport proteins. The methods were applied to two proteins from Escherichia coli: a nucleoside transporter, NupC, and a glucuronide transporter, GusB. A substantial signal for the binding of methyl [1-13 C]-Dglucuronide to GusB overexpressed in native membranes was achieved with a sample that contained as little as 20 nmol of GusB protein. The data were fitted to yield a KD value of 4.17 mM for the labeled ligand and 0.42 mM for an unlabeled ligand, p-nitrophenyl-D-glucuronide, which displaced the labeled compound. CP-MAS was also used to measure binding of [1′-13 C]uridine to overexpressed NupC. The spectrum of NupC-enriched membranes containing [1′-13 C]uridine exhibited a large peak from substrate bound to undefined sites other than the transport site, which obscured the signal from substrate bound to NupC. In a novel application of a cross-polarization/polarization-inversion (CPPI) NMR experiment, the signal from undefined binding was eliminated by use of appropriate inversion pulse lengths. By use of CPPI in a titration experiment, a KD value of 2.6 mM was determined for uridine bound to NupC. These approaches are broadly applicable to quantifying binding of substrates, inhibitors, drugs, and antibiotics to numerous membrane proteins.
bioRxiv (Cold Spring Harbor Laboratory), Feb 4, 2023
Members of the concentrative nucleoside transporter (CNT) family of proteins mediate uptake of nu... more Members of the concentrative nucleoside transporter (CNT) family of proteins mediate uptake of nucleosides into cells driven by a cation gradient, which then enter salvage pathways for nucleic acid synthesis. In humans, they also transport hydrophilic anticancer and antiviral nucleoside analogue drugs into cells and tissues where they exert their pharmacological effects. Escherichia coli CNT NupC (400 residues) is pyrimidine-specific and driven by a proton gradient. We have used computational, biochemical, and biophysical methods to characterize evolutionary relationships, conservation of residues, structural domains, transmembrane helices, and residues involved in nucleoside binding and/or transport activity in NupC compared with those of sodium-driven Vibrio cholerae CNT (vcCNT) and human CNTs (hCNT1−3). As in the crystal structure of vcCNT, NupC appears to contain eight transmembrane-spanning α-helices. Wild-type NupC and single-cysteinecontaining mutants were tested for transport activity in energized E. coli whole cells and for binding of nucleosides in non-energized native inner membranes using novel cross-polarization magic-angle spinning solid-state nuclear magnetic resonance methods. Wild-type NupC had an apparent affinity of initial rate transport (K m app) for [ 14 C]uridine of 22.2 ± 3.7 μM and an apparent binding affinity (K d app) for [1′-13 C]uridine of 1.8−2.6 mM. Mutant S142C retained transport and binding affinities similar to those of the wild type. Mutants G146C and E149C had no transport activity but retained varying degrees of partial binding activity with affinities decreasing in the following order: wild type > S142C > G146C > E149C. Results were explained with respect to a homology model of NupC based on the structure of vcCNT and a hypothetical elevator-type mechanism of alternating access membrane transport in NupC.
Molecular Membrane Biology, 2008
Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine... more Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of (13)C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly (13)C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.
Computational Biology and Chemistry, Apr 1, 2020
In this study we isolated and performed in silico analysis of a putative coclaurine N-methyltrans... more In this study we isolated and performed in silico analysis of a putative coclaurine N-methyltransferase (CNMT) from the basal angiosperm Aristolochia fimbriata. The Aristolochiaceae plant family produces alkaloids similar to the Papavaraceae family, and CNMTs are central enzymes in biosynthesis pathways producing compounds of ethnopharmacological interest. We used bioinformatics and computational tools to predict a three-dimensional homology model and to investigate the putative function of the protein and its mechanism for methylation. The putative CNMT is a unique (S)-adenosyl-L-methionine (SAM)-dependent N-methyltransferase, catalyzing transfer of a methyl group from SAM to the amino group of coclaurine. The model revealed a mixed α/β structure comprising seven twisted β-strands surrounded by twelve α-helices. Sequence comparisons and the model indicate an N-terminal catalytic Core domain and a C-terminal domain, of which the latter forms a pocket for coclaurine. An additional binding pocket for SAM is connected to the coclaurine binding pocket by a small opening. CNMT activity is proposed to follow an S N 2-type mechanism as observed for a similarly conformed enzyme. Residues predicted for the methyl transfer reaction are Tyr79 and Glu96, which are conserved in the sequence from A. fimbriata and in homologous N-methyltransferases. The isolated CNMT is the first to be investigated from any basal angiosperm.
PubMed, Nov 1, 2022
The agent responsible for the COVID-19 pandemic was the newly discovered coronavirus SARS-CoV-2. ... more The agent responsible for the COVID-19 pandemic was the newly discovered coronavirus SARS-CoV-2. A trimeric spike protein on the SARS-CoV-2 virion binds to the ACE2 receptor on host cells. In this study we performed a structure-based virtual screening and molecular docking of existing drugs against a high-resolution structure of the SARS-CoV-2 spike protein-ACE2 receptor complex. The 2.5-Å crystal structure of the C-terminal domain of the SARS-CoV-2 spike protein (residues 319-541) in complex with human ACE2 (SARS-CoV-2-S-CTD/hACE2) (PDB ID: 6LZG) was used as the target for screening 4,374 FDA-approved drugs from the ZINC15 database using PyRx software. Molecular docking was performed using BIOVIA Discovery Studio Visualizer. The top twenty highest affinity drugs had binding energies of -7.0 to -8.8 kcal/mol. The highest affinity drug was the selective vasopressin V2-receptor antagonist Tolvaptan, for which molecular docking identified drug-amino acid residue interactions with ACE2. Other drugs displaying binding energies better than -8.0 kcal/mol were Nizoral, Amaryl, Accolate, Sorafenib, Glipizide and Azelastine. The predicted interactions of these highest affinity drugs with residues in ACE2 were at positions that could disrupt the spike protein-ACE2 complex, so these drugs have the potential to be repurposed as inhibitors of the SARS-CoV-2 virus.
Journal of the Chemical Society of Pakistan, May 4, 2020
Nickel(II) complexes of the following Schiff base ligands derived from 4-(dimethylamino)benzaldeh... more Nickel(II) complexes of the following Schiff base ligands derived from 4-(dimethylamino)benzaldehyde were synthesised: (Z)-1-(4-(dimethylamino)benzylideneamino)-propan-1-ol through condensation with 1-amino-propan-1-ol, (N1E,N2E)-N1, N2-bis(4-dimethylamino)benzylidene)benzene-1,2-diamine through condensation with benzene-1,2-diamine and 2-((4-dimethylamino)benzylidene)amino)phenol through condensation with 2-aminophenol. The synthesised Schiff bases and the resultant Ni(II) complexes were all isolated as crystals and characterized by melting point, elemental analysis, infrared spectroscopy, NMR spectroscopy, mass spectrometry and by conductance measurements. Antioxidant activity of the Ni(II) complexes was tested by assay with the free radical compound 2,2-diphenyl-1-picrylhydrazyl (DPPH).
Solid-state nuclear magnetic resonance (ssNMR) spectroscopy can obtain structural, functional and... more Solid-state nuclear magnetic resonance (ssNMR) spectroscopy can obtain structural, functional and ligand-binding information about frozen and solid-like biological samples. ssNMR spectra can be enhanced by orders of magnitude using the technique of dynamic nuclear polarisation (DNP). In DNP there is polarisation transfer from high-gyromagnetic ratio (γ) unpaired electrons to neighbouring nuclei using microwave irradiation at or near the electron Larmor frequency. This produces an absolute increase in the signal-to-noise ratio and allows experiments on much smaller quantities of sample and/or using much shorter acquisition times. Along with necessary instrumentation an essential requirement for DNP-ssNMR is a sample with an endogenous free radical or an exogenous free radical polarising agent must be added to the sample. The polarising agent must be soluble in the sample matrix and compatible with the biological sample. The free radical(s) of the polarising agent also must be stable for the lifetime of DNP-ssNMR experiments. Nitroxides have been most used as polarising agents, including the biradical compounds TOTAPOL and AMUPol with a wide range of biological samples to produce DNP enhancement factors () of up to 250. Derivatives of TOTAPOL and AMUPol and many other different polarising agents have also been used. Whilst conventional polarising agents are mixed throughout the sample, others are targeted at specific sites to provide a more localised signal enhancement. Here we review the different polarising agents and spin-tags that have been used in DNP-ssNMR studies on biological samples.
Asian journal of biological and life sciences, 2018
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Molecular Membrane Biology, Nov 17, 2015
Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and ... more Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.
Molecular Membrane Biology, Aug 2, 2011
Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many... more Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all highresolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both b-barrel and a-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design.
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Papers by Simon G Patching
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.