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Yu-Chen Lee, Jenny Arnling Bååth, Ryan M Bastle, Sonali Bhattacharjee, Mary Jo Cantoria, Mark Dornan, Enrique Gamero-Estevez, Lenzie Ford, Lenka Halova, Jennifer Kernan, Charlotte Kürten, Siran Li, Jerahme Martinez, Nalani Sachan, Medoune Sarr, Xiwei Shan, Nandhitha Subramanian, Keith Rivera, Darryl Pappin, Sue-Hwa Lin
Detergents play an essential role during the isolation of membrane protein complexes. Inappropriate use of detergents may affect the native fold of the membrane proteins, their binding to antibodies, or their interaction with partner proteins. Here we used cadherin-11 (Cad11) as an example to examine the impact of detergents on membrane protein complex isolation. We found that mAb 1A5 could immunoprecipitate Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but not by octylglucoside, suggesting that octylglucoside interferes with Cad11-mAb 1A5 interaction...
January 5, 2018: Journal of Proteome Research
Rui Zhou, Guanghui Yang, Yigong Shi
γ-secretase is an intramembrane protease complex consisting of nicastrin, presenilin-1/2, APH-1a/b, and Pen-2. Hydrolysis of the 99-residue transmembrane fragment of amyloid precursor protein (APP-C99) by γ-secretase produces β-amyloid (Aβ) peptides. Pathogenic mutations in PSEN1 and PSEN2 , which encode the catalytic subunit presenilin-1/2 of γ-secretase, lead to familial Alzheimer's disease in an autosomal dominant manner. However, the underlying mechanism of how the mutant PSEN gene may affect the function of the WT allele remains to be elucidated...
November 28, 2017: Proceedings of the National Academy of Sciences of the United States of America
K S Mineev, K D Nadezhdin, S A Goncharuk, A S Arseniev
Structural studies of membrane proteins are of great importance and interest, with solution and solid state NMR spectroscopy being very promising tools for that task. However, such investigations are hindered by a number of obstacles, and in the first place by the fact that membrane proteins need an adequate environment that models the cell membrane. One of the most widely used and prospective membrane mimetics is isotropic bicelles. While large anisotropic bicelles are well-studied, the field of small bicelles contains a lot of "white spots"...
July 5, 2016: Langmuir: the ACS Journal of Surfaces and Colloids
Christina J Crump, Heather E Murrey, T Eric Ballard, Christopher W Am Ende, Xianzhong Wu, Natalya Gertsik, Douglas S Johnson, Yue-Ming Li
γ-Secretase is a multiprotein complex that catalyzes intramembrane proteolysis associated with Alzheimer's disease and cancer. Here, we have developed potent sulfonamide clickable photoaffinity probes that target γ-secretase in vitro and in cells by incorporating various photoreactive groups and walking the clickable alkyne handle to different positions around the molecule. We found that benzophenone is preferred over diazirine as a photoreactive group within the sulfonamide scaffold for labeling γ-secretase...
August 17, 2016: ACS Chemical Neuroscience
Luis F Padilla-Morales, José O Colón-Sáez, Joel E González-Nieves, Orestes Quesada-González, José A Lasalde-Dominicci
In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC)...
January 2016: Biochimica et Biophysica Acta
Ramon Barnadas-Rodríguez, Josep Cladera
CHAPSO and CHAPS are zwitterionic surfactants derived from bile salts which are usually employed in protein purification and for the preparation of liposomes and bicelles. Despite their spread use, there are significant discrepancies on the critical concentrations that determine their aggregation behavior. In this work, we study the interaction between these surfactants with the negative fluorescent dye pyranine (HPTS) by absorbance, fluorescence, and infrared spectrometry to establish their concentration-dependent aggregation...
August 25, 2015: Langmuir: the ACS Journal of Surfaces and Colloids
Wilaiwan Sriwimol, Aratee Aroonkesorn, Somsri Sakdee, Chalermpol Kanchanawarin, Takayuki Uchihashi, Toshio Ando, Chanan Angsuthanasombat
The insecticidal feature of the three-domain Cry δ-endotoxins from Bacillus thuringiensis is generally attributed to their capability to form oligomeric pores, causing lysis of target larval midgut cells. However, the molecular description of their oligomerization process has not been clearly defined. Here a stable prepore of the 65-kDa trypsin-activated Cry4Ba mosquito-specific toxin was established through membrane-mimetic environments by forming an ∼200-kDa octyl-β-D-glucoside micelle-induced trimer...
August 21, 2015: Journal of Biological Chemistry
Martin Textor, Sandro Keller
Determination of the critical micellar concentration of surfactants and of the heat of demicellization by means of isothermal titration calorimetry usually involves either calculation of the first derivative of the heat of demicellization with respect to surfactant concentration or application of a generic sigmoidal fit to the demicellization isotherm. Here, we show that a combination of both approaches provides an unbiased and reproducible data analysis strategy without the need for user input other than the calorimetric data proper...
September 15, 2015: Analytical Biochemistry
Kimberly Stephens, Catherine D Thaler, Richard A Cardullo
For successful fertilization to occur, molecules on the surface of male and female gametes must recognize each other in a complementary manner. In some organisms, sperm possess a glycosidase on the plasma membrane overlying the head while eggs have glycoproteins that are recognized by those glycosidases resulting in sperm-egg recognition. In this study, two glycosidases, mannosidase and β-N-acetylglucosaminidase, were identified and biochemically characterized in Aquarius remigis sperm. The mannosidase had a Km of 2...
May 2015: Insect Biochemistry and Molecular Biology
Hannah Hazel Morales, Qasim Saleem, Peter M Macdonald
The properties of bicelles composed of mixtures of long-chain lipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG), stabilized by zwitterionic bile salt analogue 3-[(3-cholamidopropyl)dimethyl-d6-ammonio]-2-hydroxy-1-propanesulfonate (CHAPSO-d6), deuterated at both amino methyls, were investigated by a combination of (31)P and (2)H NMR, focusing on the behavior of CHAPSO as a function of temperature. For compositions of molar ratio q = [DMPC + DMPG]/[CHAPSO] = 3, R = [DMPG]/[DMPC + DMPG] = 0, 0...
December 23, 2014: Langmuir: the ACS Journal of Surfaces and Colloids
LiYing Zhao, Hiroyuki Sakagami, Tatsuo Suzuki
We systematically investigated the purification process of post-synaptic density (PSD) and post-synaptic membrane rafts (PSRs) from the rat forebrain synaptic plasma membranes by examining the components and the structures of the materials obtained after the treatment of synaptic plasma membranes with TX-100, n-octyl β-d-glucoside (OG) or 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO). These three detergents exhibited distinct separation profiles for the synaptic subdomains. Type I and type II PSD proteins displayed mutually exclusive distribution...
October 2014: Journal of Neurochemistry
James A Anstead, Steven D Hartson, Gary A Thompson
BACKGROUND: The transport of sugars, hormones, amino acids, proteins, sugar alcohols, and other organic compounds from the sites of synthesis to the sites of use or storage occurs through the conducting cells of the phloem. To better understand these processes a comprehensive understanding of the proteins involved is required. While a considerable amount of data has been obtained from proteomic analyses of phloem sap, this has mainly served to identify the soluble proteins that are translocated through the phloem network...
2013: BMC Genomics
Samreen Amani, Faisal Nasim, Taqi Ahmed Khan, Naveed Ahmad Fazili, Mohammad Furkan, Imtiyaz Ahmad Bhat, Javed Masood Khan, Rizwan Hasan Khan, Aabgeena Naeem
Role of micellar environment created by Triton X-100 (TX-100) and CHAPSO on protein conformation using IgG as a model system has been studied in this paper. A substantial amount of secondary structure with the reduction in constant tertiary contacts was obtained in both bovine and human IgG in the presence of 0.12 mM TX-100 where as 6 and 8 mM CHAPSO concentration was required for this type of secondary structure. Further addition of either of the detergents result in the induction of α-helix in both the IgGs as evident by helix specific peaks in the amide I region of FTIR and circular dichroism spectra...
2014: Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
Ming Li, Hannah H Morales, John Katsaras, Norbert Kučerka, Yongkun Yang, Peter M Macdonald, Mu-Ping Nieh
Spontaneously forming structures of a system composed of dimyristoyl phosphatidylcholine (DMPC) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) were studied by small-angle neutron scattering (SANS), (31)P NMR, and stimulated echo (STE) pulsed field gradient (PFG) (1)H NMR diffusion measurements. Charged lipid dimyristoyl phosphatidylglycerol (DMPG) was used to induce different surface charge densities. The structures adopted were investigated as a function of temperature and lipid concentration for samples with a constant molar ratio of long-chain to short-chain lipids (= 3)...
December 23, 2013: Langmuir: the ACS Journal of Surfaces and Colloids
Rachna Ujwal, Jeff Abramson
Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. MPs have substantial hydrophobic regions embedded within the bilayer...
2012: Journal of Visualized Experiments: JoVE
Hui Wang, Johannes Elferich, Eric Gouaux
Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-β-D-glucopyranoside (β-OG) and the number of substrate binding sites are controversial issues...
February 2012: Nature Structural & Molecular Biology
Ritchie Williamson, Andrew J Thompson, Mika Abu, Abdul Hye, Alessia Usardi, Steven Lynham, Brian H Anderton, Diane P Hanger
BACKGROUND: Membrane rafts are small highly dynamic sterol- and sphingolipid-enriched membrane domains that have received considerable attention due to their role in diverse cellular functions. More recently the involvement of membrane rafts in neuronal processes has been highlighted since these specialized membrane domains have been shown to be involved in synapse formation, neuronal polarity and neurodegeneration. Detergent resistance followed by gradient centrifugation is often used as first step in screening putative membrane raft components...
September 22, 2010: BMC Neuroscience
L Vian, J Vincent, J Maurin, I Fabre, J Giroux, J P Cano
Three in vitro cytotoxicity assays [neutral red uptake assay (NRU), MTT test and total protein content determination (TPC)] were analysed to assess their value for predicting the ocular irritancy potential of 20 surfactants. For each test, three established cell lines (SIRC rabbit corneal cells, Balb/c 3T3 and L929 mouse fibroblasts) were used. The concentration that induced 50% inhibition relative to controls (IC(50)) was calculated for each test, cell line and chemical. In vivo ocular irritancy data were compared with in vitro results...
April 1995: Toxicology in Vitro: An International Journal Published in Association with BIBRA
Yasser Qutub, Veselina Uzunova, Oleg Galkin, Peter G Vekilov
To address the interactions of hemin with phospholipid bilayers, we introduce hemin to a solution of dimyristoylphosphatidylcholine (DMPC), a long chain phospholipid, and 3-(cholamidopropyl)(dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), a detergent, in which vesicles form at around 37 degrees C. We show that vesicles composed of DMPC/CHAPSO form and grow, following a mechanism that does not trap solution and excludes larger solutes, such as hemin, from the vesicle interior. The existence of a limited number of patches of likely 2D crystalline hemin embedded in the phospholipid bilayer suggests that this layer is saturated with hemin molecules...
April 8, 2010: Journal of Physical Chemistry. B
Mako Takami, Yu Nagashima, Yoshihisa Sano, Seiko Ishihara, Maho Morishima-Kawashima, Satoru Funamoto, Yasuo Ihara
Amyloid beta protein (Abeta), a pathogenic molecule associated with Alzheimer's disease, is produced by gamma-secretase, which cleaves the beta-carboxyl terminal fragment (betaCTF) of beta-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that betaCTF is cleaved first at the membrane-cytoplasm boundary, producing two long Abetas, Abeta(48) and Abeta(49), which are processed further by releasing three residues at each step to produce Abeta(42) and Abeta(40), respectively...
October 14, 2009: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
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