Search results for "Glycophorin"
showing 10 items of 17 documents
Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase
2020
Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplas…
Mcl-1 and Bok transmembrane domains : Unexpected players in the modulation of apoptosis
2020
The Bcl-2 protein family comprises both proand antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family mem-bers can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate p…
The role of hydrophobic matching on transmembrane helix packing in cells
2017
Folding and packing of membrane proteins are highly influenced by the lipidic component of the membrane. Here, we explore how the hydrophobic mismatch (the difference between the hydrophobic span of a transmembrane protein region and the hydrophobic thickness of the lipid membrane around the protein) influences transmembrane helix packing in a cellular environment. Using a ToxRED assay in Escherichia coli and a Bimolecular Fluorescent Complementation approach in human-derived cells complemented by atomistic molecular dynamics simulations we analyzed the dimerization of Glycophorin A derived transmembrane segments. We concluded that, biological membranes can accommodate transmembrane homo-di…
Detergent Properties Influence the Stability of the Glycophorin A Transmembrane Helix Dimer in Lysophosphatidylcholine Micelles
2012
AbstractDetergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents w…
Unfolding a transmembrane helix dimer: A FRET study in mixed micelles
2009
The exact nature of membrane protein folding and assembly is not understood in detail yet. Addition of SDS to a membrane protein dissolved in mild, non-polar detergent results in formation of mixed micelles and in subsequent denaturation of higher ordered membrane protein structures. The exact nature of this denaturation event is, however, enigmatic, and separation of an individual helix pair in mixed micelles has also not been reported yet. Here we followed unfolding of the human glycophorin A transmembrane helix dimer in mixed micelles by fluorescence spectroscopy. Energy transfer between differently labelled glycophorin A transmembrane helices decreased with increasing SDS mole fractions…
Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro.
2009
The pComb3H vector system is used for constructing and panning recombinant antibody libraries. It allows for expression of monovalent Fab fragments, either on the surface of M13 phage, or in the form of soluble proteins secreted into the periplasmic space of bacteria. We constructed a modified pComb3H vector containing cDNA encoding for a 23-amino acid fragment of the Escherichia coli biotin carboxy carrier protein (BCCP), which is an acceptor sequence for biotinylation. The vector was used to express the Fab fragment recognizing human glycophorin A. The purified Fab fragment containing this biotin acceptor sequence was effectively biotinylated in vitro using biotin ligase (BirA). The speci…
AN IL-6/IL-6 SOLUBLE RECEPTOR (IL-6R) HYBRID PROTEIN (H-IL-6) INDUCES EPO-INDEPENDENT ERYTHROID DIFFERENTIATION IN HUMAN CD34+CELLS
2000
H-IL-6 is a hybrid protein constructed to contain IL-6 and its soluble receptor linked by a flexible peptide chain. Here we show that H-IL-6 strongly enhances proliferation of human CD34(+)cells in serum-free liquid culture, and that the majority of the cells generated belong to the erythroid lineage, being positive for the marker Glycophorin A. Conversely, H-IL-6 does not increase the number of myeloid, CD13-positive cells. Comparable effects are observed on progenitors from cord blood and adult peripheral blood. Therefore, H-IL-6 triggers an erythroid-inducing signal in haematopoietic progenitor cells, independently from erythropoietin (EPO).
Heme Binding Constricts the Conformational Dynamics of the Cytochrome b559′ Heme Binding Cavity
2012
Cytochrome b(559)' is a transmembrane protein formed by homodimerization of the 44-residue PsbF polypeptide and noncovalent binding of a heme cofactor. The PsbF polypeptide can dimerize in the absence and presence of heme. To monitor structural alterations associated with binding of heme to the apo-cytochrome, we analyzed the apo- and holo-cytochrome structure by electron paramagnetic resonance spectroscopy. Spin labeling of amino acids located close to the heme binding domain of the cytochrome revealed that the structure of the heme binding domain is unconstrained in the absence of heme. Heme binding restricts the conformational dynamics of the heme binding domain, resulting in the structu…
Influence of proline residues in transmembrane helix packing
2003
Integral membrane proteins often contain proline residues in their alpha-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In additi…
Influence of the C-terminus of the glycophorin A transmembrane fragment on the dimerization process
2000
The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane‐mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C‐terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement o…