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A pH-Mediated Topological Switch within the N-Terminal Domain of Human Caveolin-3.
Kim JH, Schlebach JP, Lu Z, Peng D, Reasoner KC, Sanders CR
(2016) Biophys J 110: 2475-2485
MeSH Terms: Amino Acid Sequence, Caveolin 3, Circular Dichroism, Humans, Hydrogen-Ion Concentration, Membranes, Artificial, Micelles, Models, Molecular, Mutation, Nuclear Magnetic Resonance, Biomolecular, Phosphatidylglycerols, Protein Structure, Secondary, Solubility, Solutions
Show Abstract · Added November 21, 2018
Caveolins mediate the formation of caveolae, which are small omega-shaped membrane invaginations involved in a variety of cellular processes. There are three caveolin isoforms, the third of which (Cav3) is expressed in smooth and skeletal muscles. Mutations in Cav3 cause a variety of human muscular diseases. In this work, we characterize the secondary structure, dynamics, and topology of the monomeric form of the full-length lipidated protein. Cav3 consists of a series of membrane-embedded or surface-associated helical elements connected by extramembrane connecting loops or disordered domains. Our results also reveal that the N-terminal domain undergoes a large scale pH-mediated topological rearrangement between soluble and membrane-anchored forms. Considering that roughly one-third of pathogenic mutations in Cav3 influence charged residues located in this domain, we hypothesize that this transition is likely to be relevant to the molecular basis of Cav3-linked diseases. These results provide insight into the structure of Cav3 and set the stage for mechanistic investigations of the effects of pathogenic mutations.
Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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Structural investigation of the transmembrane domain of KCNE1 in proteoliposomes.
Sahu ID, Kroncke BM, Zhang R, Dunagan MM, Smith HJ, Craig A, McCarrick RM, Sanders CR, Lorigan GA
(2014) Biochemistry 53: 6392-401
MeSH Terms: Amino Acid Substitution, Humans, Lipid Bilayers, Liposomes, Molecular Dynamics Simulation, Phosphatidylcholines, Phosphatidylglycerols, Potassium Channels, Voltage-Gated, Protein Structure, Secondary, Protein Structure, Tertiary, Thermodynamics
Show Abstract · Added November 21, 2018
KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel.
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High-resolution structures and orientations of antimicrobial peptides piscidin 1 and piscidin 3 in fluid bilayers reveal tilting, kinking, and bilayer immersion.
Perrin BS, Tian Y, Fu R, Grant CV, Chekmenev EY, Wieczorek WE, Dao AE, Hayden RM, Burzynski CM, Venable RM, Sharma M, Opella SJ, Pastor RW, Cotten ML
(2014) J Am Chem Soc 136: 3491-504
MeSH Terms: Antimicrobial Cationic Peptides, Fish Proteins, Hydrophobic and Hydrophilic Interactions, Immersion, Lipid Bilayers, Liquid Crystals, Molecular Dynamics Simulation, Phosphatidylcholines, Phosphatidylglycerols, Protein Structure, Secondary
Show Abstract · Added March 5, 2014
While antimicrobial peptides (AMPs) have been widely investigated as potential therapeutics, high-resolution structures obtained under biologically relevant conditions are lacking. Here, the high-resolution structures of the homologous 22-residue long AMPs piscidin 1 (p1) and piscidin 3 (p3) are determined in fluid-phase 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) and 1:1 phosphatidylethanolamine/phosphatidylglycerol (PE/PG) bilayers to identify molecular features important for membrane destabilization in bacterial cell membrane mimics. Structural refinement of (1)H-(15)N dipolar couplings and (15)N chemical shifts measured by oriented sample solid-state NMR and all-atom molecular dynamics (MD) simulations provide structural and orientational information of high precision and accuracy about these interfacially bound α-helical peptides. The tilt of the helical axis, τ, is between 83° and 93° with respect to the bilayer normal for all systems and analysis methods. The average azimuthal rotation, ρ, is 235°, which results in burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at G13 that delineates two helical segments characterized by a small difference in their τ angles (<10°) and significant difference in their ρ angles (~25°). Remarkably, the kink, at the end of a G(X)4G motif highly conserved among members of the piscidin family, allows p1 and p3 to adopt ρ angles that maximize their hydrophobic moments. Two structural features differentiate the more potent p1 from p3: p1 has a larger ρ angle and less N-terminal fraying. The peptides have comparable depths of insertion in PC/PG, but p3 is 1.2 Å more deeply inserted than p1 in PE/PG. In contrast to the ideal α-helical structures typically assumed in mechanistic models of AMPs, p1 and p3 adopt disrupted α-helical backbones that correct for differences in the amphipathicity of their N- and C-ends, and their centers of mass lie ~1.2-3.6 Å below the plane defined by the C2 atoms of the lipid acyl chains.
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10 MeSH Terms
Reconstitution of KCNE1 into lipid bilayers: comparing the structural, dynamic, and activity differences in micelle and vesicle environments.
Coey AT, Sahu ID, Gunasekera TS, Troxel KR, Hawn JM, Swartz MS, Wickenheiser MR, Reid RJ, Welch RC, Vanoye CG, Kang C, Sanders CR, Lorigan GA
(2011) Biochemistry 50: 10851-9
MeSH Terms: Animals, Cell Membrane, Circular Dichroism, Electron Spin Resonance Spectroscopy, Humans, Lipid Bilayers, Liposomes, Micelles, Microinjections, Mutagenesis, Site-Directed, Mutant Proteins, Oocytes, Phosphatidylcholines, Phosphatidylglycerols, Potassium Channels, Voltage-Gated, Protein Stability, Protein Structure, Secondary, Recombinant Proteins, Xenopus laevis
Show Abstract · Added November 21, 2018
KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.
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Lysophospholipid micelles sustain the stability and catalytic activity of diacylglycerol kinase in the absence of lipids.
Koehler J, Sulistijo ES, Sakakura M, Kim HJ, Ellis CD, Sanders CR
(2010) Biochemistry 49: 7089-99
MeSH Terms: Circular Dichroism, Diacylglycerol Kinase, Escherichia coli, Kinetics, Lysophosphatidylcholines, Lysophospholipids, Micelles, Nuclear Magnetic Resonance, Biomolecular, Phosphatidylglycerols, Phosphorylcholine, Temperature
Show Abstract · Added August 9, 2013
There has been a renewal of interest in interactions of membrane proteins with detergents and lipids, sparked both by recent results that illuminate the structural details of these interactions and also by the realization that some experimental membrane protein structures are distorted by detergent-protein interactions. The integral membrane enzyme diacylglycerol kinase (DAGK) has long been thought to require the presence of lipid as an obligate "cofactor" in order to be catalytically viable in micelles. Here, we report that near-optimal catalytic properties are observed for DAGK in micelles composed of lysomyristoylphosphatidylcholine (LMPC), with significant activity also being observed in micelles composed of lysomyristoylphosphatidylglycerol and tetradecylphosphocholine. All three of these detergents were also sustained high stability of the enzyme. NMR measurements revealed significant differences in DAGK-detergent interactions involving LMPC micelles versus micelles composed of dodecylphosphocholine. These results highlight the fact that some integral membrane proteins can maintain native-like properties in lipid-free detergent micelles and also suggest that C(14)-based detergents may be worthy of more widespread use in studies of membrane proteins.
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11 MeSH Terms
Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum.
Murphy SC, Fernandez-Pol S, Chung PH, Prasanna Murthy SN, Milne SB, Salomao M, Brown HA, Lomasney JW, Mohandas N, Haldar K
(2007) Blood 110: 2132-9
MeSH Terms: Animals, Annexin A5, Blotting, Western, Cell Membrane, Cytoplasm, Endocytosis, Erythrocyte Membrane, Erythrocytes, Flow Cytometry, Green Fluorescent Proteins, Humans, Isoenzymes, Liposomes, Malaria, Mass Spectrometry, Membrane Microdomains, Parasitemia, Phosphatidylethanolamines, Phosphatidylglycerols, Phosphatidylinositol 4,5-Diphosphate, Phosphatidylinositols, Phospholipase C delta, Plasmodium falciparum, Primaquine, Type C Phospholipases, Vacuoles
Show Abstract · Added March 21, 2013
Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non-receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP(2)) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP(2), although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection.
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26 MeSH Terms
Analysis of RF heating and sample stability in aligned static solid-state NMR spectroscopy.
Li C, Mo Y, Hu J, Chekmenev E, Tian C, Gao FP, Fu R, Gor'kov P, Brey W, Cross TA
(2006) J Magn Reson 180: 51-7
MeSH Terms: Hot Temperature, Lipid Bilayers, Nuclear Magnetic Resonance, Biomolecular, Phosphatidylcholines, Phosphatidylglycerols, Radio Waves, Sodium Chloride, Temperature
Show Abstract · Added March 5, 2014
Sample instability during solid-state NMR experiments frequently arises due to RF heating in aligned samples of hydrated lipid bilayers. A new, simple approach for estimating sample temperature is used to show that, at 9.4 T, sample heating depends mostly on (1)H decoupling power rather than on (15)N irradiation in PISEMA experiments. Such heating for different sample preparations, including lipid composition, salt concentration and hydration level was assessed and the hydration level was found to be the primary parameter correlated with sample heating. The contribution to RF heating from the dielectric loss appears to be dominant under our experimental conditions. The heat generated by a single scan was approximately calculated from the Q values of the probe, to be a 1.7 degrees C elevation per single pulse sequence iteration under typical sample conditions. The steady-state sample temperature during PISEMA experiments can be estimated based on the method presented here, which correlates the loss factor with the temperature rise induced by the RF heating of the sample.
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8 MeSH Terms
Studies on the endogenous phospholipids of mammalian kidney and their in vitro hydrolysis by endogenous phospholipases: a thin layer chromatographic and densitometric study.
Helmy FM, Hassanein M, Juracka A
(2004) Cell Biochem Funct 22: 379-87
MeSH Terms: Animals, Cardiolipins, Cats, Chromatography, Thin Layer, Densitometry, Dogs, Guinea Pigs, Hydrolysis, Kidney, Mammals, Mice, Phosphatidylglycerols, Phospholipases, Rats
Show Abstract · Added May 29, 2014
The phosphoglycerides profile of six species of mammalian kidney (guinea pig, pig, cat, dog, mouse and rat) and their in vitro response to the endogenous phospholipases were determined by TLC technology in conjunction with densitometric measurements. Changes in their phospholipids profile subsequent to in vitro incubation of whole tissue homogenate of these kidneys for 60 min, at pH 7.4, 38 degrees C, and prior to phospholipids extraction have shown that the deacylation of the endogenous cardiolipin (CL) is the most prevalent lipolytic event of all mammalian kidneys studied. Concurrent with the deacylation of CL, there was also formation of monolysocardiolipin (MLCL) and a reduction in CL level. To a much lesser extent, lyso alkenyl phosphatidyl ethanolamine (LPE) was also produced concomitant with a decrease of the endogenous alkenyl phosphatidyl ethanolamine (PE) level. The deacylation of PE plasmalogen to its lyso form confirms the action of endogenous PLA(2) releasing sn-2 fatty acids.
Copyright (c) 2004 John Wiley & Sons, Ltd.
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14 MeSH Terms
Pulmonary surfactant metabolism in infants lacking surfactant protein B.
Beers MF, Hamvas A, Moxley MA, Gonzales LW, Guttentag SH, Solarin KO, Longmore WJ, Nogee LM, Ballard PL
(2000) Am J Respir Cell Mol Biol 22: 380-91
MeSH Terms: Acetates, Blotting, Western, Cysteine, Fetus, Gene Expression, Genotype, Humans, Infant, Infant, Newborn, Methionine, Phosphatidylcholines, Phosphatidylglycerols, Proteolipids, Pulmonary Surfactant-Associated Proteins, Pulmonary Surfactants, RNA, Messenger, Respiratory Distress Syndrome, Newborn, Sulfur Radioisotopes, Transcription, Genetic, Tritium
Show Abstract · Added January 20, 2015
Infants with inherited deficiency of pulmonary surfactant protein (SP) B develop respiratory failure at birth and die without lung transplantation. We examined aspects of surfactant metabolism in lung tissue and lavage fluid acquired at transplantation or postmortem from ten infants born at term with inherited deficiency of SP-B; comparison groups were infants with other forms of chronic lung disease (CLD) and normal infants. In pulse/chase labeling studies with cultured deficient tissue, no immunoprecipitable SP-B was observed and an approximately 6-kD form of SP-C accumulated that was only transiently present in CLD tissue. SP-B messenger RNA (mRNA) was approximately 8% of normal in deficient specimens, and some intact message was observed after, but not before, explant culture. Transcription rates for SP-B, assessed by nuclear run-on assay using probes for sequences both 5' and 3' of the common nonsense mutation (121ins2), were comparable in all lungs examined. The minimal surface tension achieved with lavage surfactant was similarly elevated in both deficient and CLD infants (26-31 mN/m) compared with normal infants (6 mN/m). Both SP-B-deficient and CLD infants had markedly decreased phosphatidylglycerol content of lavage and tissue compared with normal lung, whereas synthetic rates for phospholipids, including phosphatidylglycerol, were normal. We conclude that the mutated SP-B gene is transcribed normally but produces an unstable mRNA and that absence of SP-B protein blocks processing of SP-C. Chronic infant lung disease, of various etiologies, reduces surfactant function and apparently alters phosphatidylglycerol degradation.
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20 MeSH Terms
Utilization of heavy-atom effect quenching of pyrene fluorescence to determine the intramembrane distribution of halothane.
Washington K, Sarasua MM, Koehler LS, Koehler KA, Schultz JA, Pedersen LG, Hiskey RG
(1984) Photochem Photobiol 40: 693-701
MeSH Terms: Chloroform, Halothane, Liposomes, Phosphatidylglycerols, Pulmonary Surfactants, Pyrenes, Spectrometry, Fluorescence
Added April 12, 2016
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7 MeSH Terms