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Proceedings of the National Academy of... Feb 1990We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA...
We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In all cases, this Z-DNA binding was strongly blocked by competition with acidic phospholipids, such as cardiolipin. Our results raise the question whether many of the Z-DNA binding proteins previously isolated are actually phospholipid-binding proteins.
Topics: Carrier Proteins; Chromatography, Affinity; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Kinetics; Membrane Proteins; Molecular Weight; Nucleic Acid Conformation; Phospholipids; Polydeoxyribonucleotides
PubMed: 2406717
DOI: 10.1073/pnas.87.4.1292 -
Analytical Chemistry Aug 2020Phospholipids are important to cellular function and are a vital structural component of plasma and organelle membranes. These membranes isolate the cell from its...
Phospholipids are important to cellular function and are a vital structural component of plasma and organelle membranes. These membranes isolate the cell from its environment, allow regulation of the internal concentrations of ions and small molecules, and host diverse types of membrane proteins. It remains extremely challenging to identify specific membrane protein-lipid interactions and their relative strengths. Native mass spectrometry, an intrinsically gas-phase method, has recently been demonstrated as a promising tool for identifying endogenous protein-lipid interactions. However, to what extent the identified interactions reflect solution- versus gas-phase binding strengths is not known. Here, the "Extended" Kinetic Method and computations at three different levels of theory are used to experimentally and theoretically determine intrinsic gas-phase basicities (GB, Δ for deprotonation of the protonated base) and proton affinities (PA, Δ for deprotonation of the protonated base) of six lipids representing common phospholipid types. Gas-phase acidities (Δ and Δ for deprotonation) of neutral phospholipids are also evaluated computationally and ranked experimentally. Intriguingly, it is found that two of these phospholipids, sphingomyelin and phosphatidylcholine, have the highest GB of any small, monomeric biomolecules measured to date and are more basic than arginine. Phosphatidylethanolamine and phosphatidylserine are found to be similar in GB to basic amino acids lysine and histidine, and phosphatidic acid and phosphatidylglycerol are the least basic of the six lipid types studied, though still more basic than alanine. Kinetic Method experiments and theory show that the gas-phase acidities of these phospholipids are high but less extreme than their GB values, with phosphatidylserine and phosphatidylglycerol being the most acidic. These results indicate that sphingomyelin and phosphatidylcholine lipids can act as charge-reducing agents when dissociated from native membrane protein-lipid complexes in the gas phase and provide a straightforward model to explain the results of several recent native mass spectrometry studies of protein-lipid complexes.
Topics: Computer Simulation; Gases; Kinetics; Models, Chemical; Models, Molecular; Molecular Structure; Phospholipids; Thermodynamics
PubMed: 32628014
DOI: 10.1021/acs.analchem.0c00613 -
European Journal of Biochemistry Mar 1994The nonspecific lipid-transfer protein (nsL-TP) from bovine liver was studied by measuring the binding and transfer of the fluorescent phospholipid...
The nonspecific lipid-transfer protein (nsL-TP) from bovine liver was studied by measuring the binding and transfer of the fluorescent phospholipid 1-palmitoyl-2-[6-(1-pyrenyl)-hexanoyl]-sn-glycero-3-phosphocholine (PamPryGroPCho). A kinetic model is presented involving three steps: (a) interaction of nsL-TP with a membrane surface; (b) equilibration of PamPyrGroPCho monomers between the membrane and nsL-TP; and (c) dissociation of the nsL-TP/PamPyrGroPCho complex from the membrane surface. Steady-state analysis of the model yielded theoretical equations describing both binding and transfer kinetics. Computer analysis, using these equations, showed good fits with the experimental results and several kinetic constants could be calculated. From these constants it was inferred that incorporation of acidic phospholipids into vesicles enhanced the interaction of nsL-TP with the membrane interface (step a), without affecting the equilibrium binding of phospholipid monomers to nsL-TP (step b). As a result, the rate of nsL-TP-mediated PamPyrGroPCho transfer from donor to acceptor vesicles was greatly affected. Under the conditions of incubation, incorporation of the acidic lipids in the donor membrane vesicles stimulated transfer, whereas incorporation of these lipids in the acceptor membranes could lead to a virtually complete inhibition of transfer. From the results it is concluded that the formation of a soluble lipid-nsL-TP complex is the key step in nsL-TP-mediated phospholipid transfer.
Topics: Animals; Carrier Proteins; Cattle; Kinetics; Liposomes; Liver; Membrane Lipids; Phospholipids; Plant Proteins; Sterols; Surface Properties
PubMed: 8143718
DOI: 10.1111/j.1432-1033.1994.tb18707.x -
Biochemical and Biophysical Research... Apr 2021Phospholipid transfer protein, ∼80 kDa, transfers phospholipids from micelles to lipid binding proteins. The acceptor protein in plasma is apolipoprotein-A1, 28 kDa....
Phospholipid transfer protein, ∼80 kDa, transfers phospholipids from micelles to lipid binding proteins. The acceptor protein in plasma is apolipoprotein-A1, 28 kDa. Previously, phospholipid transfer protein was found in tears but an acceptor protein was not identified. To search for the acceptor protein(s) in tears a fluorescent phospholipid transfer assay was altered to omit the extrinsic acceptor. Human tears were incubated with fluorescent micelles and showed marked transfer activity verifying a native acceptor protein must be present. Reconstituted tears without tear lipocalin (lipocalin-1) eliminated the transfer of phospholipids. To determine if phospholipid transfer protein is involved in carrying phospholipid to the surface of tears from tear lipocalin, a fraction enriched in phospholipid transfer protein was injected into the subphase of a tear mimicking buffer in which tear lipocalin was present. The addition of phospholipid transfer protein did not increase the thickness of the surface layer regardless of the presence of lipid bearing tear lipocalin. The data show that phospholipid transfer protein transfers phospholipid from micelles to tear lipocalin. Phospholipid transfer protein does not transport the phospholipid. While tear lipocalin has no intrinsic transfer activity from micelles, it is the acceptor protein for phospholipid transfer protein in tears.
Topics: Humans; Lipocalin 1; Phospholipid Transfer Proteins; Phospholipids; Reference Standards; Spectrometry, Fluorescence; Tears; Young Adult
PubMed: 33631671
DOI: 10.1016/j.bbrc.2021.02.054 -
MBio Feb 2017Membrane deformation by proteins is a universal phenomenon that has been studied extensively in eukaryotes but much less in prokaryotes. In this study, we discovered a...
Membrane deformation by proteins is a universal phenomenon that has been studied extensively in eukaryotes but much less in prokaryotes. In this study, we discovered a membrane-deforming activity of the phospholipid -methyltransferase PmtA from the plant-pathogenic bacterium PmtA catalyzes the successive three-step -methylation of phosphatidylethanolamine to phosphatidylcholine. Here, we defined the lipid and protein requirements for the membrane-remodeling activity of PmtA by a combination of transmission electron microscopy and liposome interaction studies. Dependent on the lipid composition, PmtA changes the shape of spherical liposomes either into filaments or small vesicles. Upon overproduction of PmtA in , vesicle-like structures occur in the cytoplasm, dependent on the presence of the anionic lipid cardiolipin. The N-terminal lipid-binding α-helix (αA) is involved in membrane deformation by PmtA. Two functionally distinct and spatially separated regions in αA can be distinguished. Anionic interactions by positively charged amino acids on one face of the helix are responsible for membrane recruitment of the enzyme. The opposite hydrophobic face of the helix is required for membrane remodeling, presumably by shallow insertion into the lipid bilayer. The ability to alter the morphology of biological membranes is known for a small number of some bacterial proteins. Our study adds the phospholipid -methyltransferase PmtA as a new member to the category of bacterial membrane-remodeling proteins. A combination of and methods reveals the molecular requirements for membrane deformation at the protein and phospholipid level. The dual functionality of PmtA suggests a contribution of membrane biosynthesis enzymes to the complex morphology of bacterial membranes.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Catalysis; Cell Membrane; Liposomes; Methyltransferases; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Protein Binding
PubMed: 28196959
DOI: 10.1128/mBio.02082-16 -
The Journal of Biological Chemistry May 1990Photoaffinity labeling of calcineurin by 1,2-distearoyl-sn-glycero-3-phospho-N-(4-azido-3-[125I]iodo-2- hydroxybenzoyl)ethanolamine resulted in preferential labeling of...
Photoaffinity labeling of calcineurin by 1,2-distearoyl-sn-glycero-3-phospho-N-(4-azido-3-[125I]iodo-2- hydroxybenzoyl)ethanolamine resulted in preferential labeling of its regulatory B subunit. Photolabeling of B was greatly enhanced by Ca2+ which further supports the hypothesis that the phospholipid-binding site of calcineurin is located on this Ca2(+)-binding subunit. Extending the time of incubation of calcineurin with the photoprobe prior to photolysis also elevated labeling of the B subunit, probably as a result of time-dependent changes in protein conformation. Support for these conformational changes was obtained when time-dependent preincubation of calcineurin with acidic phospholipids enhanced subsequent tryptic degradation of its B subunit. Activity measurements and analyses of the reversibility of phospholipid-binding provided evidence for a two-stage mechanism of calcineurin-phospholipid interactions. Initial binding of calcineurin to phospholipids is rapid, Ca2(+)-sensitive, reversible, and leads to stimulation of the phosphatase toward a number of its substrates. A subsequent slow phase strengthens the association and appears to correlate with the phospholipid-promoted conformational change of the B subunit; the corresponding time-dependent effects on enzymatic activity are, again, substrate-dependent.
Topics: Affinity Labels; Animals; Azides; Brain; Calcineurin; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cattle; Enzyme Activation; Kinetics; Macromolecular Substances; Nickel; Phosphatidylethanolamines; Phospholipids; Phosphoprotein Phosphatases; Protein Binding; Substrate Specificity
PubMed: 2159005
DOI: No ID Found -
Biophysical Journal Jan 2019Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and...
Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.
Topics: Neutron Diffraction; Phosphatidylcholines; Phosphatidylinositols; Phospholipid Transfer Proteins; Protein Binding; Saccharomyces cerevisiae Proteins; Scattering, Small Angle; Unilamellar Liposomes
PubMed: 30580923
DOI: 10.1016/j.bpj.2018.11.3131 -
The Journal of Biological Chemistry Jun 1995
Review
Topics: Animals; GTP-Binding Proteins; Lysophospholipids; Phospholipids; Signal Transduction; Structure-Activity Relationship
PubMed: 7768880
DOI: 10.1074/jbc.270.22.12949 -
The Journal of Biological Chemistry May 2018The proportion of phosphatidylcholine (PC) in the membrane is controlled by CTP:phosphocholine cytidylyltransferase α (CCTα), which is known to be regulated by a dual...
The proportion of phosphatidylcholine (PC) in the membrane is controlled by CTP:phosphocholine cytidylyltransferase α (CCTα), which is known to be regulated by a dual auto-inhibitory and membrane-binding domain. However, the detailed mechanism by which this domain regulates CCTα activity is not clear. Ramezanpour use a combined computational and biochemical approach to define new details of this mechanism, providing an elegant illustration of how the lipid-sensing domain of a phospholipid biosynthetic enzyme controls membrane homeostasis.
Topics: Choline-Phosphate Cytidylyltransferase; Cytidine Triphosphate; Phosphatidylcholines; Phospholipids; Phosphorylcholine
PubMed: 29728535
DOI: 10.1074/jbc.H118.002882 -
International Journal of Molecular... Jan 2013The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane... (Review)
Review
The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane - monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro.
Topics: Lipid Bilayers; Liposomes; Membrane Proteins; Models, Chemical; Models, Molecular; Nanostructures; Phospholipids; Protein Binding; Protein Structure, Tertiary; Unilamellar Liposomes
PubMed: 23344058
DOI: 10.3390/ijms14011589