Identification and partial purification of ABGP205, an integral membrane glycoprotein from brain that binds ankyrin.
Ontology highlight
ABSTRACT:
SUBMITTER: Treharne KJ
PROVIDER: S-EPMC1149304 | biostudies-other | 1988 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Fatty acid desaturase enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids, and are divided into soluble and integral membrane classes. Crystal structures of soluble desaturases are available; however, membrane desaturases have defied decades of efforts due largely to the difficulty of generating recombinant desaturase proteins for crystallographic analysis. Mortierella alpina is an oleaginous fungus which possesses eight membrane desaturases involved in the synthesis of saturated, monounsaturated and polyunsaturated fatty acids. Here, we describe the successful expression, purification and enzymatic assay of three M. alpina desaturases (FADS15, FADS12, and FADS9-I). Estimated yields of desaturases with purity >95% are approximately 3.5% (Ca. 4.6 mg/L of culture) for FADS15, 2.3% (Ca. 2.5 mg/L of culture) for FADS12 and 10.7% (Ca. 37.5 mg/L of culture) for FADS9-I. Successful expression of high amounts of recombinant proteins represents a critical step towards the structural elucidation of membrane fatty acid desaturases.
| S-EPMC3592867 | biostudies-literature
Project description:Transmembrane proteins are greatly underrepresented in data generated by imaging mass spectrometry (IMS) because of analytical challenges related to their size and solubility. Here, we present the first example of MALDI IMS of two highly modified multitransmembrane domain proteins, myelin proteolipid protein (PLP, 30 kDa) and DM-20 (26 kDa), from various regions of rat brain, namely, the cerebrum, cerebellum, and medulla. We utilize a novel tissue pretreatment aimed at transmembrane protein enrichment to show the in situ distribution of fatty acylation of these proteins, particularly of post-translational palmitoylation. Additionally, we demonstrate the utility of protease-encapsulated hydrogels for spatially localized on-tissue protein digestion and peptide extraction for subsequent direct coupling to LC-MS/MS for protein identification.
| S-EPMC3782084 | biostudies-literature
Project description:We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions.
| S-EPMC1189342 | biostudies-literature
Project description:Protein-lipid interactions are increasingly recognized as central to the structure and function of membrane proteins. However, with the exception of simplified models, specific protein-lipid interactions are particularly difficult to highlight experimentally. Here, we used molecular dynamics simulations to identify a specific protein-lipid interaction in lactose permease, a prototypical model for transmembrane proteins. The interactions can be correlated with the functional dependence of the protein to specific lipid species. The technique is simple and widely applicable to other membrane proteins, and a variety of lipid matrices can be used.
| S-EPMC2856259 | biostudies-literature
Project description:G-protein-coupled receptors (GPCRs) and other structurally and functionally related membrane proteins represent particularly attractive targets for drug discovery. Integral membrane proteins are often difficult to purify from native contexts, and lack of sufficient quantities hampers subsequent structural and functional proteomic studies. We describe here an optimized enrichment strategy involving a membrane protein-compatible 1D4 affinity tag that is derived from the carboxy-terminal nine amino residues of bovine rhodopsin, and its corresponding tag-specific, high-affinity monoclonal antibody. When two GPCRs as well as two related ATP binding cassette (ABC) transporters are expressed in their functional forms in human cell lines, we have shown that a single detergent and wash condition can be employed for the purification of all said membrane proteins. Subsequent in-gel digestion with trypsin and mass spectrometric peptide analysis resulted in high sequence coverage for the ABC transporters ABCA1-1D4 and ABCA4-1D4. In contrast, digestion by various enzymatic combinations was necessary to obtain the best sequence coverage for affinity-enriched GPCRs CXCR4-1D4 and CCR5-1D4 as compared against other entries in an annotated spectrum library. Furthermore, specific enzyme combinations were necessary to produce suitable peptides for deducing N-glycosylation sites on CXCR4. Our results demonstrate that the 1D4-tag enrichment strategy is a versatile tool for the characterization of integral membrane proteins that can be employed for functional proteomic studies.
| S-EPMC4103948 | biostudies-literature
Project description:The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This 'solubilization by design' approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.
| S-EPMC3565690 | biostudies-literature
Project description:The complete primary structure of an integral membrane glycoprotein of the nuclear pore was deduced from the cDNA sequence. The cDNA encodes a polypeptide of 204,205 D containing a 25-residue-long signal sequence, two hydrophobic segments that could function as transmembrane segments, and 13 potential N-linked oligosaccharide addition sites. Endoglycosidase H reduces the molecular mass by approximately 9 kD suggesting that not all of these 13 sites are used. We discuss possible models for the topology of this protein in the pore membrane as well as a possible role in the formation of pores and pore complexes.
| S-EPMC2115607 | biostudies-other
Project description:Classical swine fever (CSF) or hog cholera, caused by a positive stranded RNA virus belonging to the genus Pestivirus of the Flaviviridae family, is highly contagious and fatal disease of pigs. We report the novel design of construct for production of highly soluble glycoprotein Erns fragment using prokaryotic expression system. A truncated fragment of the Erns gene (coding for aa 109-170) denoted as 'Erns-Ag' was subcloned and expressed as hexa-histidine tag fusion on both terminus of protein in Escherichia coli. The highly soluble recombinant Erns-Ag protein with purity >95 % was purified by one step Ni-NTA affinity chromatography under native condition. Anti Erns-Ag polyclonal antibodies raised in guinea pig was found to react with CSFV antigen in infected MDCK cell line during immunoperoxidase test. The described methodology of producing a highly soluble recombinant protein with native conformation would likely to assist in development of differential diagnostic test as well as its application in raising hyperimmune sera for detection of CSFV antigen either in tissue materials or infected cell lines.
| S-EPMC3550794 | biostudies-literature
Project description:OMA1 is a mitochondrial protease. Among its substrates are DELE1, a signaling peptide, which can elicit the integrated stress response, as well as the membrane-shaping dynamin-related GTPase OPA1, which can drive mitochondrial outer membrane permeabilization. OMA1 is dormant under physiological conditions but rapidly activated upon mitochondrial stress, such as loss of membrane potential or excessive reactive oxygen species. Accordingly, OMA1 was found to be activated in a number of disease conditions, including cancer and neurodegeneration. OMA1 has a predicted transmembrane domain and is believed to be tethered to the mitochondrial inner membrane. Yet, its structure has not been resolved and its context-dependent regulation remains obscure. Here, I review the literature with focus on OMA1's biochemistry. I provide a good homology model of OMA1's active site with a root-mean-square deviation of 0.9 Å and a DALI Z-score of 19.8. And I build a case for OMA1 actually being an integral membrane protease based on OMA1's role in the generation of small signaling peptides, its functional overlap with PARL, and OMA1's homology with ZMPSTE24. The refined understanding of this important enzyme can help with the design of tool compounds and development of chemical probes in the future.
| S-EPMC7770061 | biostudies-literature
Project description:The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.
| S-EPMC3448790 | biostudies-literature