Secondary literature sources for DUF1944
The following references were automatically generated.
- Garewal M, Zhang L, Ren G
- Optimized negative-staining protocol for examining lipid-protein interactions by electron microscopy.
- Methods Mol Biol. 2013; 974: 111-8
- Display abstract
A large number of proteins are capable of inserting themselves into lipids, and interacting with membranes, such as transmembrane proteins and apolipoproteins. Protein-lipid interactions have been identified as one of the keys in understanding biological processes, while the structure of proteins at the lipid-binding stage can provide evidence to help identify their roles and critical functions. However, structure determination of proteins at the lipid-binding stage is rather difficult, because conformational and compositional heterogeneities of the protein-lipid complexes are major barriers to unravel their structures using traditional methods, such as X-ray crystallography. Electron microscopy (EM) is an alternative approach to determine protein structure and has demonstrated a capability in visualizing lipid-protein interactions directly. Among various EM techniques, negative-staining (NS) is an easy, rapid, qualitative approach that is a well-established technique, frequently used in research laboratories. Conventional NS protocols, unfortunately, often generate artifacts with lipid-related proteins, such as the rouleau formation of lipoproteins. To overcome this artifact formation, Ren and his colleagues recently developed an optimized NS protocol that was validated by comparing images of lipoproteins from cryo-electron microscopy (cryo-EM). The optimized NS protocol could produce "near native-state" particle images and high contrast images of the protein in its lipid-binding state that is favorable for three-dimensional (3D) reconstruction by single-particle analysis and individual-particle electron tomography (IPET), suggesting this optimized protocol can be used widely to examine the structure of proteins at lipid-binding stage.
- Grossfield A
- Special issue on lipid-protein interactions.
- Chem Phys Lipids. 2013; 169: 1-1
- Dyer DH, Vyazunova I, Lorch JM, Forest KT, Lan Q
- Characterization of the yellow fever mosquito sterol carrier protein-2 like 3 gene and ligand-bound protein structure.
- Mol Cell Biochem. 2009; 326: 67-77
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The sterol carrier protein-2 like 3 gene (AeSCP-2L3), a new member of the SCP-2 protein family, is identified from the yellow fever mosquito, Aedes aegypti. The predicted molecular weight of AeSCP-2L3 is 13.4 kDa with a calculated pI of 4.98. AeSCP-2L3 transcription occurs in the larval feeding stages and the mRNA levels decrease in pupae and adults. The highest levels of AeSCP-2L3 gene expression are found in the body wall, and possibly originated in the fat body. This is the first report of a mosquito SCP-2-like protein with prominent expression in tissue other than the midgut. The X-ray protein crystal structure of AeSCP-2L3 reveals a bound C16 fatty acid whose acyl tail penetrates deeply into a hydrophobic cavity. Interestingly, the ligand-binding cavity is slightly larger than previously described for AeSCP-2 (Dyer et al. J Biol Chem 278:39085-39091, 2003) and AeSCP-2L2 (Dyer et al. J Lipid Res M700460-JLR200, 2007). There are also an additional 10 amino acids in SCP-2L3 that are not present in other characterized mosquito SCP-2s forming an extended loop between beta 3 and beta 4. Otherwise, the protein backbone is exceedingly similar to other SCP-2 and SCP-2-like proteins. In contrast to this observed high structural homology of members in the mosquito SCP2 family, the amino acid sequence identity between the members is less than 30%. The results from structural analysis imply that there have been evolutionary constraints that favor the SCP-2 C(alpha) backbone fold while the specificity of ligand binding can be altered.
- Sippel KH et al.
- Structure determination of the cancer-associated Mycoplasma hyorhinis protein Mh-p37.
- Acta Crystallogr D Biol Crystallogr. 2008; 64: 1172-8
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The crystal structure of the Mycoplasma hyorhinis protein Mh-p37 has been solved and refined to 1.9 A resolution. This is the first de novo structure to be determined using the recently described heavy-atom reagent [Beck et al. (2008), Acta Cryst. D64, 1179-1182] 5-amino-2,4,6-triiodoisophthalic acid (I3C), which contains three I atoms arranged in an equilateral triangle, by SIRAS methods. Data collection was performed in-house at room temperature. SHELXD and SHELXE were used to determine the I-atom positions and phase the native protein and PHENIX AutoBuild software was used to automatically fit the amino-acid sequence to the electron-density map. The structure was refined using SHELX97 to an R(cryst) of 18.6% and an R(free) of 24.0%. Mh-p37 is an alpha/beta protein with two well defined domains which are separated by a deep cleft. An unanticipated ligand bound in the center of the molecule at the base of the cleft has been modeled as thiamine pyrophosphate or vitamin B(1). Retrospective attempts to solve the crystal structure by Patterson search methods using either isomorphous or anomalous differences failed. Additionally, attempts to use proteins with the highest structural homology in the Protein Data Bank to phase the data by molecular replacement were unsuccessful, most likely in hindsight because of their poor structural agreement. Therefore, the I3C reagent offers an alternative, quick and inexpensive method for in-house phasing of de novo structures where other methods may not be successful.
- Jolivet P, Boulard C, Chardot T, Anton M
- New insights into the structure of apolipoprotein B from low-density lipoproteins and identification of a novel YGP-like protein in hen egg yolk.
- J Agric Food Chem. 2008; 56: 5871-9
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Apoproteins of low-density lipoproteins (LDL) and soluble proteins (livetins) contained in hen egg yolk plasma have been demonstrated as being essential to the interfacial and emulsifying properties of yolk. The knowledge of their structure is necessary to better understand these properties. Purified protein fractions were separated by SDS-PAGE or 2D-PAGE and identified through the LC-MS/MS of their trypsin peptides. Hen blood apolipoprotein B gives rise to nine different apoproteins in LDL after maturation and proteolysis. Among these apoproteins, two protein fragments appeared to be less accessible to proteases and could be enriched in beta-sheets and firmly associated with lipids. Plasma soluble proteins were constituted by approximately 45% of yolk immunoglobulins with a high heterogeneity of the variable regions of both heavy and light chains, 41% of glycoproteins constituted by YGP42 and YGP40, 14% of albumins, and one new minor protein we called YGP30, showing 75% similarity to YGP40.
- Griffin MD et al.
- Phospholipid interaction induces molecular-level polymorphism in apolipoprotein C-II amyloid fibrils via alternative assembly pathways.
- J Mol Biol. 2008; 375: 240-56
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A common feature of many of the most important and prominent amyloid-forming proteins is their ability to bind lipids and lipid complexes. Lipids are ubiquitous components of disease-associated amyloid plaques and deposits in humans, yet the specific roles of lipid in the process of amyloid fibril formation are poorly understood. This study investigated the effect of phospholipids on amyloid fibril formation by human apolipoprotein (apo) C-II using phosphatidylcholine derivatives comprising acyl chains of up to 14 carbon atoms. Submicellar concentrations of short-chain phospholipids increase the rate of apoC-II fibril formation in an acyl-chain-length- and concentration-dependent fashion, while high micellar concentrations of phospholipids completely inhibited amyloid formation. At lower concentrations of soluble phospholipid complexes, fibril formation by apoC-II was only partially inhibited, and under these conditions, aggregation followed a two-phase process. Electron microscopy showed that the fibrils resulting from the second phase of aggregation were straight, cablelike, and about 13 nm wide, in contrast to the homogeneous twisted-ribbon morphology of apoC-II fibrils formed under lipid-free conditions. Seeding experiments showed that this alternative fibril structure could be templated both in the presence and in the absence of lipid complex, suggesting that the two morphologies result from distinct assembly pathways. Circular dichroism spectroscopy studies indicated that the secondary structural conformation within the straight-type and ribbon-type fibrils were distinct, further suggesting divergent assembly pathways. These studies show that phospholipid complexes can change the structural architecture of mature fibrils and generate new fibril morphologies with the potential to alter the in vivo behaviour of amyloid. Such lipid interactions may play a role in defining the structural features of fibrils formed by diverse amyloidogenic proteins.
- Jones MR
- Lipids in photosynthetic reaction centres: structural roles and functional holes.
- Prog Lipid Res. 2007; 46: 56-87
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Photosynthetic proteins power the biosphere. Reaction centres, light harvesting antenna proteins and cytochrome b(6)f (or bc(1)) complexes are expressed at high levels, have been subjected to an intensive spectroscopic, biochemical and mutagenic analysis, and several have been characterised to an informatively high resolution by X-ray crystallography. In addition to revealing the structural basis for the transduction of light energy, X-ray crystallography has brought molecular insights into the relationships between these multicomponent membrane proteins and their lipid environment. Lipids resolved in the X-ray crystal structures of photosynthetic proteins bind light harvesting cofactors, fill intra-protein cavities through which quinones can diffuse, form an important part of the monomer-monomer interface in multimeric structures and may facilitate structural flexibility in complexes that undergo partial disassembly and repair. It has been proposed that individual lipids influence the biophysical properties of reaction centre cofactors, and so affect the rate of electron transfer through the complex. Lipids have also been shown to be important for successful crystallisation of photosynthetic proteins. Comparison of the three types of reaction centre that have been structurally characterised reveals interesting similarities in the position of bound lipids that may point towards a generic requirement to reinforce the structure of the core electron transfer domain. The crystallographic data are also providing new opportunities to find molecular explanations for observed effects of different types of lipid on the structure, mechanism and organisation of reaction centres and other photosynthetic proteins.
- Dashti N, Manchekar M, Liu Y, Sun Z, Segrest JP
- Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells.
- J Biol Chem. 2007; 282: 28597-608
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We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. (2004) J. Biol. Chem. 279, 39757-39766). In this study we have investigated the putative functional role of MTP in the initial lipidation of apoB:1000 in stable transformants of McA-RH7777 cells. Inhibition of MTP lipid transfer activity by 0.1 microm BMS-197636 and 5, 10, and 20 microm of BMS-200150 had no detectable effect on the synthesis, lipidation, and secretion of apoB:1000-containing particles. Under identical experimental conditions, the synthesis, lipidation, and secretion of endogenous apoB100-containing particles in HepG2 and parental untransfected McA-RH7777 cells were inhibited by 86-94%. BMS-200150 at 40 microm nearly abolished the secretion of endogenous apoB100-containing particles in HepG2 and parental McA-RH cells but caused only 15-20% inhibition in the secretion of apoB: 1000-containing particles. This modest decrease was attributable to the nonspecific effect of a high concentration of this compound on hepatic protein synthesis, as reflected in a similar (20-25%) reduction in albumin secretion. Suppression of MTP gene expression in stable transformants of McA-RH7777 cells by micro-interfering RNA led to 60-70% decrease in MTP mRNA and protein levels, but it had no detectable effect on the secretion of apoB:1000. Our results provide a compelling argument that the initial addition of phospholipids to apoB:1000 and initiation of apoB-containing lipoprotein assembly occur independently of MTP lipid transfer activity.
- Isoe J, Hagedorn HH
- Mosquito vitellogenin genes: Comparative sequence analysis, gene duplication, and the role of rare synonymous codon usage in regulating expression.
- J Insect Sci. 2007; 7: 1-49
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Comparative sequence analysis of mosquito vitellogenin (Vg) genes was carried out to gain a better understanding of their evolution. The genomic clones of vitellogenin genes were isolated and sequenced from all three subfamilies of the family Culicidae including Culicinae (Aedes aegypti, Ochlerotatus atropalpus, Ae. polynesiensis, Ae. albopictus, Ochlerotatus triseriatus and Culex quinquefasciatus), Toxorhynchitinae (Toxorhynchites amboinensis), and Anophelinae (Anopheles albimanus). Genomic clones of vitellogenin genes Vg-B and Vg-C were isolated from Ae. aegypti and sequenced. A comparison of Vg-B and Vg-C, with the previously characterized vitellogenin gene, Vg-A1, suggests that Vg-A1 and Vg-B probably arose by a recent gene duplication, and Vg-C apparently diverged from the two other members of the gene family in an earlier gene duplication event. Two vitellogenin genes orthologous to Vg-C were cloned from a Cx. quinquefasciatus DNA library, one of which is truncated at the N-terminal end. Single vitellogenin genes, orthologous to Vg-C, were cloned from the An. albimanus and Tx. amboinensis libraries. Incomplete sequences orthologous to Vg-B and Vg-C were isolated from the Oc. atropalpus library. Only partial sequences were isolated from Ae. polynesiensis, Ae. albopictus and Oc. triseriatus. Inferred phylogenetic relationships based on analysis of these sequences suggest that Vg-C was the ancestral gene and that a recent gene duplication gave rise to Vg-A1 and Vg-B after the separation of the genus Aedes. The deduced amino acid composition of mosquito vitellogenin proteins exhibits higher tyrosine and phenylalanine composition than other mosquito proteins except for the hexamerin storage proteins. Analysis of vitellogenin coding sequences showed that a majority of amino acid substitutions were due to conserved and moderately conserved changes suggesting that the vitellogenins are under moderately selective constrains to maintain tertiary structure. The vitellogenin genes of the three anautogenous mosquitoes, that require a blood meal to develop eggs, had very high synonymous codon usage biases similar to highly expressed genes of other organisms. On the other hand, the vitellogenin genes of autogenous mosquitoes, that develop at least one batch of eggs without a blood meal, exhibited low synonymous codon usage bias. An unusual pattern of synonymous codon usage was observed in the first 15 amino acid residues encoding the signal peptide in the vitellogenin genes, where a high number of rarely used synonymous codons are present. It is hypothesized that rare synonymous codons have selectively accumulated in the signal peptide region to down-regulate the rate of translation initiation in the absence of a blood meal. Real-time PCR gene expression experiments showed that all three Ae. aegypti vitellogenin genes were highly expressed after a blood meal, and expressed in non-blood-fed females, males, larvae and pupae at trace levels. Sequences were deposited in GenBank (accession numbers: Ae. aegypti Vg-B, AY380797, Vg-C, AY373377; Oc. atropalpus Vg-B, AY691321, Vg-C, AY691322; Ae. polynesiensis Vg-A1, AY691318, Vg-B, AY691319, Vg-C, AY691320; Ae. albopictus Vg-A1, AY691316, Vg-C, AY691317; Oc. triseriatus Vg-C, AY691323; Cx. quinquefasciatus Vg-C1, AY691324, Vg-C2, AY691325; Tx. amboinensis Vg-C, AY691326; An. albimanus Vg-C, AY691327).
- Trostchansky A, Rubbo H
- Lipid nitration and formation of lipid-protein adducts: biological insights.
- Amino Acids. 2007; 32: 517-22
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Lipid-protein adducts are formed during oxidative and nitrative stress conditions associated with increasing lipid and protein oxidation and nitration. The focus of this review is the analysis of interactions between oxidative-modified lipids and proteins and how lipid nitration can modulate lipid-protein adducts formation. For this, two biologically-relevant models will be analysed: a) human low density lipoprotein, whose oxidation is involved in the early steps of atherogenesis, and b) alpha-synuclein/lipid membranes system, where lipid-protein adducts are being associated with the develop of Parkinson disease and other synucleinopathies.
- Gunasekaran K, Nussinov R
- How different are structurally flexible and rigid binding sites? Sequence and structural features discriminating proteins that do and do not undergo conformational change upon ligand binding.
- J Mol Biol. 2007; 365: 257-73
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Proteins are dynamic molecules and often undergo conformational change upon ligand binding. It is widely accepted that flexible loop regions have a critical functional role in enzymes. Lack of consideration of binding site flexibility has led to failures in predicting protein functions and in successfully docking ligands with protein receptors. Here we address the question: which sequence and structural features distinguish the structurally flexible and rigid binding sites? We analyze high-resolution crystal structures of ligand bound (holo) and free (apo) forms of 41 proteins where no conformational change takes place upon ligand binding, 35 examples with moderate conformational change, and 22 cases where a large conformational change has been observed. We find that the number of residue-residue contacts observed per-residue (contact density) does not distinguish flexible and rigid binding sites, suggesting a role for specific interactions and amino acids in modulating the conformational changes. Examination of hydrogen bonding and hydrophobic interactions reveals that cases that do not undergo conformational change have high polar interactions constituting the binding pockets. Intriguingly, the large, aromatic amino acid tryptophan has a high propensity to occur at the binding sites of examples where a large conformational change has been noted. Further, in large conformational change examples, hydrophobic-hydrophobic, aromatic-aromatic, and hydrophobic-polar residue pair interactions are dominant. Further analysis of the Ramachandran dihedral angles (phi, psi) reveals that the residues adopting disallowed conformations are found in both rigid and flexible cases. More importantly, the binding site residues adopting disallowed conformations clustered narrowly into two specific regions of the L-Ala Ramachandran map. Examination of the dihedral angles changes upon ligand binding shows that the magnitude of phi, psi changes are in general minimal, although some large changes particularly between right-handed alpha-helical and extended conformations are seen. Our work further provides an account of conformational changes in the dihedral angles space. The findings reported here are expected to assist in providing a framework for predicting protein-ligand complexes and for template-based prediction of protein function.
- Rava P, Ojakian GK, Shelness GS, Hussain MM
- Phospholipid transfer activity of microsomal triacylglycerol transfer protein is sufficient for the assembly and secretion of apolipoprotein B lipoproteins.
- J Biol Chem. 2006; 281: 11019-27
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Human microsomal triacylglycerol transfer protein (hMTP) is essential for apolipoprotein B (apoB)-lipoprotein assembly and secretion and is known to transfer triacylglycerols, cholesterol esters, and phospholipids. To understand the relative importance of each lipid transfer activity, we compared the ability of hMTP and its Drosophila ortholog (dMTP) to assemble apoB lipoproteins and to transfer various lipids. apoB48 secretion was induced when co-expressed with either hMTP or dMTP in COS cells, and oleic acid supplementation further augmented secretion without altering particle density. C-terminal epitope-tagged dMTP (dMTP-FLAG) facilitated the secretion of apoB polypeptides in the range of apoB48 to apoB72 but was approximately 50% as efficient as hMTP-FLAG. Comparison of lipid transfer activities revealed that although phospholipid transfer was similar in both orthologs, dMTP was unable to transfer neutral lipids. We conclude that the phospholipid transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB lipoproteins and may represent its earliest function evolved for the mobilization of lipid in invertebrates. Identification of MTP inhibitors, which selectively affect transfer of a specific lipid class, may have therapeutic potential.
- Walker A, Ando S, Smith GD, Lee RF
- The utilization of lipovitellin during blue crab (Callinectes sapidus) embryogenesis.
- Comp Biochem Physiol B Biochem Mol Biol. 2006; 143: 201-8
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Embryos of the blue crab Callinectes sapidus develop in egg sacs carried on the abdomen of the female. They develop over a period of 10-13 days at 28 degrees C and are nutritionally dependent on yolk until they emerge from the egg sacs as free-swimming zoeae. The principal component of blue crab yolk is lipovitellin (LpII), a water-soluble lipoprotein composed of approximately equal amounts of lipid and protein. We followed changes in the concentration of apoproteins of LpII during embryogenesis by ELISA and Western blots, using monoclonal antibodies against two LpII apoprotein associated peptides identified as Protein A (107 kDa) and Protein B (75 kDa). During embryogenesis there was a decrease in Protein B but an increase in two smaller peptides (52 and 35 kDa) that reacted with the Protein B antibody. Utilization of LpII during embryogenesis was also followed morphologically by immunohistochemistry. Utilization of LpII was slow in early embryonic stages, followed by rapid utilization in late embryonic stages, such that only traces of LpII were present at the end of embryogenesis. The cells of the developing hepatopancreas appear to play an important role in the utilization of LpII.
- Unwin N
- Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
- J Mol Biol. 2005; 346: 967-89
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We present a refined model of the membrane-associated Torpedo acetylcholine (ACh) receptor at 4A resolution. An improved experimental density map was obtained from 342 electron images of helical tubes, and the refined structure was derived to an R-factor of 36.7% (R(free) 37.9%) by standard crystallographic methods, after placing the densities corresponding to a single molecule into an artificial unit cell. The agreement between experimental and calculated phases along the helical layer-lines was used to monitor progress in the refinement and to give an independent measure of the accuracy. The atomic model allowed a detailed description of the whole receptor in the closed-channel form, including the ligand-binding and intracellular domains, which have not previously been interpreted at a chemical level. We confirm that the two ligand-binding alpha subunits have a different extended conformation from the three other subunits in the closed channel, and identify several interactions on both pairs of subunit interfaces, and within the alpha subunits, which may be responsible for their "distorted" structures. The ACh-coordinating amino acid side-chains of the alpha subunits are far apart in the closed channel, indicating that a localised rearrangement, involving closure of loops B and C around the bound ACh molecule, occurs upon activation. A comparison of the structure of the alpha subunit with that of AChBP having ligand present, suggests how the localised rearrangement overcomes the distortions and initiates the rotational movements associated with opening of the channel. Both vestibules of the channel are strongly electronegative, providing a cation-stabilising environment at either entrance of the membrane pore. Access to the pore on the intracellular side is further influenced by narrow lateral windows, which would be expected to screen out electrostatically ions of the wrong charge and size.
- Jiang ZG, Carraway M, McKnight CJ
- Limited proteolysis and biophysical characterization of the lipovitellin homology region in apolipoprotein B.
- Biochemistry. 2005; 44: 1163-73
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Apolipoprotein B (apoB) is the essential nonexchangeable protein in chylomicrons and very low-density lipoprotein-derived lipoprotein particles, including low-density lipoprotein (LDL). ApoB has been a key target for cardiovascular research because of its essential role in the assembly, secretion, delivery, and receptor binding of LDL. The three-dimensional structure of apoB has not been determined. However, the N-terminal region of apoB is homologous to the lipid storage protein lipovitellin, which allows the modeling of this region based on the X-ray structure of lipovitellin. The model of the N-terminal 17% of apoB (B17) suggests that, like lipovitellin, B17 consists of an N-terminal beta-barrel domain, a helical domain, and a beta-sheet domain (C-sheet). Here we test the validity of this model by limited proteolysis of B17 and the characterization of individual domains expressed in Escherichia coli and insect cell systems that are consistent with the model and proteolysis data. Circular dichroism studies of the individual domains indicate that they are folded and their secondary structures are in agreement with the model. We find that the helical domain and C-sheet of apoB interact with each other in vitro, suggesting a strong interaction between these two domains, even without a covalent peptide bond linkage. Our data suggest that the three lipovitellin-like domains exist in B17. Furthermore, the domains fold independently with secondary structures and stabilities like those of intact B17.
- Miller CE et al.
- Neutron and X-ray scattering studies of cholera toxin interactions with lipid monolayers at the air-liquid interface.
- Colloids Surf B Biointerfaces. 2005; 40: 159-63
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Using neutron/X-ray reflectivity and X-ray grazing incidence diffraction (GID), we have characterized the structure of mixed DPPE:GM(1) lipid monolayers before and during the binding of cholera toxin (CTAB(5)) or its B subunit (CTB(5)). Structural parameters such as the density and thickness of the lipid layer, extension of the GM(1) oligosaccharide headgroup, and orientation and position of the protein upon binding are reported. Both CTAB(5) and CTB(5) were measured to have approximately 50% coverage when bound to the lipid monolayer. X-ray GID experiments show that both the lipid monolayer and the cholera toxin layer are crystalline. The effects of X-ray beam damage have been assessed and the monolayer/toxin structure does not change with time after protein binding has saturated.
- Richardson PE et al.
- Assembly of lipoprotein particles containing apolipoprotein-B: structural model for the nascent lipoprotein particle.
- Biophys J. 2005; 88: 2789-800
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Apolipoprotein B (apoB) is the major protein component of large lipoprotein particles that transport lipids and cholesterol. We have developed a detailed model of the first 1000 residues of apoB using standard sequence alignment programs (ClustalW and MACAW) and the MODELLER6 package for three-dimensional homology modeling. The validity of the apoB model was supported by conservation of disulfide bonds, location of all proline residues in turns and loops, and conservation of the hydrophobic faces of the two C-terminal amphipathic beta-sheets, betaA (residues 600-763) and betaB (residues 780-1000). This model suggests a lipid-pocket mechanism for initiation of lipoprotein particle assembly. In a previous model we suggested that microsomal triglyceride transfer protein might play a structural role in completion of the lipid pocket. We no longer think this likely, but instead propose a hairpin-bridge mechanism for lipid pocket completion. Salt-bridges between four tandem charged residues (717-720) in the turn of the hairpin-bridge and four tandem complementary residues (997-1000) at the C-terminus of the model lock the bridge in the closed position, enabling the deposition of an asymmetric bilayer within the lipid pocket.
- Molina ML, Encinar JA, Barrera FN, Fernandez-Ballester G, Riquelme G, Gonzalez-Ros JM
- Influence of C-terminal protein domains and protein-lipid interactions on tetramerization and stability of the potassium channel KcsA.
- Biochemistry. 2004; 43: 14924-31
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KcsA is a prokaryotic potassium channel formed by the assembly of four identical subunits around a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portion of KcsA is known [Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77], the identification of the molecular determinant(s) involved in promoting subunit tetramerization remains to be determined. Here, C-terminal deletion channel mutants, KcsA Delta125-160 and Delta120-160, as well as 1-125 KcsA obtained from chymotrypsin cleavage of full-length 1-160 KcsA, have been used to evaluate the role of the C-terminal segment on the stability and tetrameric assembly of the channel protein. We found that the lack of the cytoplasmic C-terminal domain of KcsA, and most critically the 120-124 sequence stretch, impairs tetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modeling of KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogen bonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once the KcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociation into subunits is not greatly influenced by whether the entire C-terminal domain continues being part of the protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminal domain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsA tetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobic interactions involving intramembrane protein domains.
- Perry A, Tambyrajah W, Grossmann JG, Lian LY, Scrutton NS
- Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase.
- Biochemistry. 2004; 43: 3167-82
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Here we provide insights into the molecular structure of the two-iron 19-kDa rubredoxin (AlkG) of Pseudomonas oleovorans using solution-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering studies. Sequence alignment and biochemical studies have suggested that AlkG comprises two rubredoxin folds connected by a linker region of approximately 70 amino acid residues. The C-terminal domain (C-Rb) of this unusual rubredoxin, together with approximately 35 amino acid residues of the predicted linker region, was expressed in Escherichia coli, purified in the one-iron form and the structure of the cadmium-substituted form determined at high-resolution by NMR spectroscopy. The structure shows that the C-Rb domain is similar in fold to the conventional one-iron rubredoxins from other organisms, whereas the linker region does not have any discernible structure. This tandem "flexible-folded" structure of the polypeptide chain derived for the C-Rb protein was confirmed using solution X-ray scattering methods. X-ray scattering studies of AlkG indicated that the 70-amino acid residue linker forms a structured, yet mobile, polypeptide segment connecting the globular N- and C-terminal domains. The X-ray scattering studies also showed that the N-terminal domain (N-Rb) has a molecular conformation similar to that of C-Rb. The restored molecular shape indicates that the folded N-Rb and C-Rb domains of AlkG are noticeably separated, suggesting some domain movement on complex formation with rubredoxin reductase to allow interdomain electron transfer between the metal centers in AlkG. This study demonstrates the advantage of combining X-ray scattering and NMR methods in structural studies of dynamic, multidomain proteins that are not suited to crystallographic analysis. The study forms a structural foundation for functional studies of the interaction and electron-transfer reactions of AlkG with rubredoxin reductase, also reported herein.
- Madani S, Frenoux JM, Prost J, Belleville J
- Changes in serum lipoprotein lipids and their fatty acid compositions and lipid peroxidation in growing rats fed soybean protein versus casein with or without cholesterol.
- Nutrition. 2004; 20: 554-63
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OBJECTIVE: We compared the effects of diets based on soybean protein and casein supplemented or not supplemented with 0.1% cholesterol on plasma lipoprotein lipid amounts and their fatty acid compositions, lecithin:cholesterol acyl-transferase activity, and lipid peroxidation. METHODS: The composition and concentration of lipid and apolipoprotein in different lipoprotein classes, plasma LCAT activity, and lipid peroxidation were determined in rats fed 20% highly purified soybean protein or casein with or without 0.1% cholesterol for 2 mo. RESULTS: Soybean protein and casein diets with or without cholesterol had similar plasma total cholesterol concentrations. Soybean protein consumption diminished very low-density lipoprotein particle number, as measured by diminished contents of very low-density lipoprotein triacylglycerol, phospholipid, and apolipoprotein-B100. Lecithin:cholesterol acyl-transferase activity was not significantly modified by either protein. The soybean protein diet decreased the linoleate desaturation index (20:4[omega-6]/18:2[omega-6]) in liver and high-density lipoprotein fraction 2-3-phospholipids but enhanced red blood cell resistance against free radical attack. Addition of cholesterol to both protein diets decreased concentrations of high-density lipoprotein fraction 2-3 cholesterol. Lecithin:cholesterol acyl-transferase activity tended to be greater after cholesterol feeding, likely due to the enhanced high-density lipoprotein fraction 2-3 apolipoprotein-AI, a cofactor activator for lecithin:cholesterol acyl-transferase. Regardless of dietary protein source, cholesterol supplementation decreased the linoleate desaturation index in liver and plasma lipoprotein lipids and red blood cell resistance to free radical attack. CONCLUSIONS: Our results suggest that the dietary protein origin affects lipid peroxidation and polyunsaturated fatty acid biosynthesis and distribution among liver and different lipoprotein lipid classes, but plays only a minor role in the regulation of plasma and lipoprotein cholesterol concentrations. Providing dietary cholesterol (0.1%) with casein or soybean protein attenuates the effects of these proteins, with the exception of plasma cholesterol.
- Antonkine ML, Jordan P, Fromme P, Krauss N, Golbeck JH, Stehlik D
- Assembly of protein subunits within the stromal ridge of photosystem I. Structural changes between unbound and sequentially PS I-bound polypeptides and correlated changes of the magnetic properties of the terminal iron sulfur clusters.
- J Mol Biol. 2003; 327: 671-97
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The X-ray structure of Photosystem I (PS I) from Synechococcus elongatus was recently solved at 2.5A resolution (PDB entry 1JB0). It provides a structural model for the stromal subunits PsaC, PsaD and PsaE, which comprise the "stromal ridge" of PS I. In a separate set of studies the three-dimensional solution structures of the unbound, recombinant PsaC (PDB entry 1K0T) and PsaE (PDB entries 1PSF, 1QP2 and 1GXI) subunits were solved by NMR. The PsaC subunit of PS I is a small (9.3 kDa) protein that harbors binding sites for two [4Fe-4S] clusters F(A) and F(B), which are the terminal electron acceptors in PS I. Comparison of the PsaC structure in solution with that in the X-ray structure of PS I reveals significant differences between them which are summarized and evaluated here. Changes in the magnetic properties of [4Fe-4S] centers F(A) and F(B) are related to changes in the protein structure of PsaC, and they are further influenced by the presence of PsaD. Based on experimental evidence, three assembly stages are analyzed: PsaC(free), PsaC(only), PsaC(PS I). Unbound, recombinant PsaD, studied by NMR, has only a few elements of secondary structure and no stable three-dimensional structure in solution. When PsaD is bound in PS I, it has a well-defined three-dimensional structure. For PsaE the three-dimensional structure is very similar in solution and in the PS I-bound form, with the exception of two loop regions. We suggest that the changes in the structures of PsaC and PsaD are caused by the sequential formation of multiple networks of contacts between the polypeptides of the stromal ridge and between those polypeptides and the PsaA/PsaB core polypeptides. The three-dimensional structure of the C(2)-symmetric F(X)-binding loops on PsaA and PsaB were also analyzed and found to be significantly different from the binding sites of other proteins that contain interpolypeptide [4Fe-4S] clusters. The aim of this work is to relate contact information to structural changes in the proteins and to propose a model for the assembly of the stromal ridge of PS I based on this analysis.
- Fukada Y
- [Regulation of protein function by lipid modifications].
- Tanpakushitsu Kakusan Koso. 1999; 44: 1319-20
- Montorzi M, Falchuk KH, Vallee BL
- Vitellogenin and lipovitellin: zinc proteins of Xenopus laevis oocytes.
- Biochemistry. 1995; 34: 10851-8
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Xenopus laevis vitellogenin is a plasma protein that contains a total of 5 mol of metal/440 kDa dimer, 2 mol of zinc, and 3 mol of calcium (Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413]. There are no other group IIB or transition metals in the molecule. The zinc atoms are removed instantaneously by 1,10-phenanthroline (OP) (pK 4.8). Once internalized by receptor-mediated endocytosis, vitellogenin is cleaved into multiple polypeptides, i.e., the two lipovitellin subunits (1 and 2) plus phosvitin; these are then stored as microcrystals within yolk platelets. We here show by metal analysis of the individual proteins generated by vitellogenin processing that zinc and calcium occur in different domains of the vitellogenin polypeptide chain. All of the vitellogenin zinc is present in lipovitellin, in amounts equal to 1 mol of zinc/141 kDa. Calcium, in contrast, is detected exclusively in phosvitin which, in addition, contains 3 mol of magnesium/35 kDa, apparently acquired following vitellogenin entry into the oocyte. The zinc in lipovitellin is removed by OP in a concentration-dependent manner with a pK of 4.8, identical to that obtained for vitellogenin, and by exposure to acidic conditions (below pH 5). Following removal of zinc, the two lipovitellin subunits remain associated, suggesting that zinc is not involved in their interaction. On exposure to 1% SDS, lipovitellin does dissociate into 106 and 33 kDa subunits. The presence of stoichiometric quantities of zinc in both vitellogenin and lipovitellin calls for the study of the hitherto unrecognized biochemistry and functions of these proteins in zinc metabolism and development of the frog oocyte and embryo.
- Timmins PA, Poliks B, Banaszak L
- The location of bound lipid in the lipovitellin complex.
- Science. 1992; 257: 652-5
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The location of the bound lipid in the soluble lipoprotein lipovitellin has been determined by neutron crystallographic techniques. With the use of the contrast variation method, whereby the crystals are soaked in different H2O-D2O mixtures, the lipid has been found to occupy a large cavity in the protein whose structure had previously been determined by x-ray crystallography. The lipid appears to be bound in the form of a bilayer with the major protein-lipid interactions being hydrophobic and with the lipid headgroups projecting into the bulk solvent and into a solvent-filled space in the cavity.
- Fretheim K, Sleigh RW, Burley RW
- Formation of complexes between lecithin and apovitellenin I, an avian egg-yolk apoprotein.
- Lipids. 1986; 21: 127-31
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In a study of lipid-protein interactions in egg yolk, it was found that L-alpha-dipalmitoyl lecithin gave two distinct noncovalent complexes (A and B) with apovitellenin I, an apoprotein in the major yolk lipoprotein. Interaction took place under widely varied conditions, and yolk lecithin gave similar complexes. Complex A, which was formed within minutes, consisted of round particles of about 9 nm diameter. Complex B, which was formed more slowly, consisted of larger particles, possibly resembling curved discs, with diameter of 30-40 nm. The preparation and some properties of these complexes are described. It is suggested that they may be suitable for an extensive study of phospholipid-protein interactions in yolk.
- Raag R, Roderick S, Banaszak L
- A comparison of negatively stained electron micrographs and projections obtained from single crystal X-ray studies: the lipovitellin complex from lamprey.
- J Ultrastruct Mol Struct Res. 1986; 94: 77-84
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The analysis of single crystals of the lipovitellin complex from lamprey has made it possible to compare electron density projections derived from X-ray diffraction and density modification methods with previously published electron micrographs. The close correlation between the images obtained by the two methods demonstrates that the fidelity of images obtained by electron microscopy is excellent despite the loss of specimen order. These correlations also attest to the ability of density modification methods, currently used in macromolecular crystallography, to estimate the phases of small angle X-ray reflections.
- Ross J, Wrenn RF, Ohlendorf DH, Banaszak LJ
- Lipid domains in the yolk lipoprotein complex.
- Ann N Y Acad Sci. 1980; 348: 408-18
- Meslar HW, White HB 3rd
- Preparation of lipid-free protein extracts of egg yolk.
- Anal Biochem. 1978; 91: 75-81
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1-Butanol extraction of chicken egg yolk homogenates containing 1 M NaCl yields lipid-free aqueous solutions of egg yolk proteins. These solutions, after dialysis, can be applied to a variety of chromatographic media without clogging. Although some proteins are denatured by this procedure, most of the water-soluble proteins remain in solution, including biotin-binding protein and riboflavin-binding protein.
- MITKOV V, TARALOV S
- CHANGES OF PROTEIN AND LIPID METABOLISM IN CEREBRAL VASCULAR DISEASES.
- Folia Med (Plovdiv). 1964; 6: 169-73