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Peter Müller

Publications and source records attributed to Peter Müller.

At least 37 records · Page 2Linked to original sources
PubMed2005

ATP induces conformational changes of periplasmic loop regions of the maltose ATP-binding cassette transporter.

We have studied cofactor-induced conformational changes of the maltose ATP-binding cassette transporter by employing limited proteolysis in detergent solution. The transport complex consists of one copy each of the transmembrane subunits, MalF and MalG, and of two copies of the nucleotide-binding subunit, MalK. Transport activity further requires the periplasmic maltose-binding protein, MalE. Binding of ATP to the MalK subunits increased the susceptibility of two tryptic cleavage sites in the periplasmic loops P2 of MalF and P1 of MalG, respectively. Lys(262) of MalF and Arg(73) of MalG were identified as probable cleavage sites, resulting in two N-terminal peptide fragments of 29 and 8 kDa, respectively. Trapping the complex in the transition state by vanadate further stabilized the fragments. In contrast, the tryptic cleavage profile of MalK remained largely unchanged. ATP-induced conformational changes of MalF-P2 and MalG-P1 were supported by fluorescence spectroscopy of complex variants labeled with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. The results suggest that complex variants exhibiting a binding protein-independent phenotype (MalF500) or containing a mutation that affects the "catalytic carboxylate" (MalKE159Q) reside in a transition state-like conformation. A similar conclusion was drawn for a complex containing a replacement of MalKQ140 in the signature sequence by leucine, whereas substitution of lysine for Gln(140) appears to lock the transport complex in the ground state. Together, our data provide the first evidence for conformational changes of the transmembrane subunits of an ATP-binding cassette import system upon binding of ATP.

ATP-Binding Cassette Transporters↗
PubMed2005

Local heat flux and energy loss in a two-dimensional vibrated granular gas.

We performed event-driven simulations of a two-dimensional granular gas between two vibrating walls and directly measured the local heat flux and local energy dissipation in the stationary state. Describing the local heat flux as a function of the coordinate in the direction perpendicular to the driving walls, we test a generalization of Fourier's law, q(x)=-kappa inverted delta T(x)+mu inverted delta rho(x), by relating the local heat flux to the local gradients of the temperature and density. This ansatz accounts for the fact that heat flux can also be generated by density gradients, not only by temperature gradients. Assuming the transport coefficients kappa and mu to be independent of x, we check the validity of this assumption and test the generalized Fourier law in the simulations. Both kappa and mu are determined for different system parameters, in particular, for a wide range of coefficients of restitution. We also compare our numerical results to existing hydrodynamic theories. Agreement is found for kappa for very small inelasticities only, i.e., when the gradients are small. Beyond this region, kappa and mu exhibit a striking nonmonotonic behavior. This may hint that hydrodynamics to Navier-Stokes order cannot be applied to moderately inelastic vibrated systems.

Journal Article↗
PubMed2005

StarD10, a START domain protein overexpressed in breast cancer, functions as a phospholipid transfer protein.

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.

Animals↗
PubMed2005

Headgroup-specific exposure of phospholipids in ABCA1-expressing cells.

ABCA1 has been established to be required for the efflux of cholesterol and phospholipids to apolipoproteins such as apoA-I. At present, it is unclear whether ABCA1-mediated lipid exposure is specific with regard to lipid headgroups and whether it requires calcium activation and the presence of a lipid acceptor. In the present work, we found exofacial exposure of endogenous phosphatidylserine in the absence of apoA-I to be enhanced in ABCA1-GFP expressing MDCKII and HeLa cells compared with control cells. By using C6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phospholipid analogues, we observed elevated redistribution of phosphatidylserine and phosphatidylethanolamine but not of phosphatidylcholine analogues from the cytoplasmic to the exoplasmic leaflet of the plasma membrane of ABCA1-GFP expressing cells. Whereas glyburide affected neither the level of exofacial endogenous PS nor the outward movement of the amino phospholipid analogues, the latter was sensitive to intracellular Ca2+ in ABCA1-GFP expressing cells, further enhancing outward analogue redistribution with respect to control cells. Both receptor-mediated endocytosis and fluidphase endocytosis were reduced in MDCKII cells expressing ABCA1-GFP. Glyburide raised the level of receptor-mediated endocytosis in the ABCA1-GFP expressing cell to the level of control cells in the absence of glyburide. In control cells, however, fluid-phase endocytosis but not receptor-mediated endocytosis was significantly reduced upon glyburide treatment.

ATP Binding Cassette Transporter 1↗
PubMed2005

The 1.70 angstroms X-ray crystal structure of Mycobacterium tuberculosis phosphoglycerate mutase.

The single-crystal X-ray structure of phosphoglycerate mutase from Mycobacterium tuberculosis has been determined at a resolution of 1.70 angstroms. The C-terminal tail of each of the subunits is flexible and disordered; however, for one of the four chains (chain A) all but five residues of the chain could be modeled. Noteworthy features of the structure include the active site and a proline-rich segment in each monomer forming a short left-handed polyprolyl helix. These segments lie on the enzyme surface and could conceivably participate in protein-protein interactions.

Amino Acid Sequence↗
PubMed2005

Rheology of gelling polymers in the Zimm model.

In order to study rheological properties of gelling systems in dilute solution, we investigate the viscosity and the normal stresses in the Zimm model [B. H. Zimm, J. Chem. Phys. 24, 269 (1956)]. for randomly cross-linked monomers. The distribution of cluster topologies and sizes is assumed to be given either by Erdos-Renyi random graphs or three-dimensional bond percolation. Within this model the critical behavior of the viscosity and of the first normal stress coefficient is determined by the power-law scaling of their averages over clusters of a given size n with n. We investigate these scaling relations numerically and conclude that the scaling exponents are independent of the hydrodynamic interaction strength. The numerically determined exponents agree well with experimental data for branched polymers. However, we show that this traditional model of polymer physics is not able to yield a critical divergence at the gel point of the viscosity for a polydisperse dilute solution of gelation clusters. A generally accepted scaling relation for the Zimm exponent of the viscosity is thereby disproved.

Journal Article↗
PubMed2005

Structural and molecular characterization of equine sperm-binding fibronectin-II module proteins.

Phospholipid-binding proteins in the male genital tract are characterized by differing numbers Fn-2 modules (B-domain) carrying N-terminal extensions (A-domain) of variable length. In the stallion, three different proteins were identified, SP-1, SP-2, and EQ-12. SP-1 and SP-2 of the AA'BB'- and ABB'-type, respectively, are major proteins of the seminal plasma. Here we report the cDNA sequences of SP-1, and of a new member of the SP-2 family (SPnew) and the partial characterization of their iso- and glycoforms. The phosphorylcholine (PC)-binding ability of the long Fn-2 protein, EQ-12, with four tandemly arranged Fn-2 modules was determined by PC-affinity chromatography. Expression patterns of EQ-12, and the SP-proteins were studied by means of RT-PCR, Northern blot analysis and immunological approaches indicating differential expression along the male reproductive tract. The vast majority of the short SP-1 and SP-2 proteins are produced by the ampulla whereas EQ-12 originates from the epididymis. Indirect immunofluorescence microscopy of sperm isolated from different regions of the epididymis and Western blot analysis indicate that both, the long and the short Fn-2 proteins associate to the sperm surface during post-testicular maturation. Sperm binding of Fn-2 proteins at the post-acrosome and midpiece was at first detected in the corpus epididymis. Enhanced fluorescence intensity after ejaculation point to an increased number of molecules bound to the sperm surface. The function of these proteins is discussed in regard to their structure-function relationships.

Amino Acid Sequence↗
PubMed2004

Desmosterol may replace cholesterol in lipid membranes.

Recently, knockout mice entirely lacking cholesterol have been described as showing only a mild phenotype. For these animals, synthesis of cholesterol was interrupted at the level of its immediate precursor, desmosterol. Since cholesterol is a major and essential constituent of mammalian cellular membranes, we asked whether cholesterol with its specific impact on membrane properties might be replaced by desmosterol. By employing various approaches of NMR, fluorescence, and EPR spectroscopy, we found that the properties of phospholipid membranes like lipid packing in the presence of cholesterol or desmosterol are very similar. However, for lanosterol, a more distant precursor of cholesterol synthesis, we found significant differences in comparison with cholesterol and desmosterol. Our results show that, from the point of view of membrane biophysics, cholesterol and desmosterol behave identically and, therefore, replacement of cholesterol by desmosterol may not impact organism homeostasis.

Cholesterol↗
PubMed2004

Local equation of state and velocity distributions of a driven granular gas.

We present event-driven simulations of a granular gas of inelastic hard disks with incomplete normal restitution in two dimensions between vibrating walls (without gravity). We measure hydrodynamic quantities such as the stress tensor, density and temperature profiles, as well as velocity distributions. Relating the local pressure to the local temperature and local density, we construct a local constitutive equation. For strong inelasticities the local constitutive relation depends on global system parameters, like the volume fraction and the aspect ratio. For moderate inelasticities the constitutive relation is approximately independent of the system parameters and can hence be regarded as a local equation of state, even though the system is highly inhomogeneous with heterogeneous temperature and density profiles arising as a consequence of energy injection. With respect to local velocity distributions we find that they do not scale with the square root of the local granular temperature. Moreover the high-velocity tails are different for the distribution of the x and the y components of the velocity, and even depend on the position in the sample, the global volume fraction, and the coefficient of restitution.

Journal Article↗
PubMed2004

Use of Tn KPK2 for sequencing a 10.6-kb PstI DNA fragment of Bradyrhizobium japonicum and for the construction of aspA and ndvA mutants.

Transposon Tn KPK2 was used to saturate a randomly cloned Bradyrhizobium japonicum PstI fragment and the insertions were used as starting points for the sequence determination. The first gene of the 10.6-kb DNA insert encodes a homologue to ndvA, the product of which is known to be involved in the formation of periplasmic cyclic glucans. Selected Tn KPK2 insertions were introduced into the B. japonicum wild-type strain. The resulting mutants were subsequently tested for their symbiotic interactions with soybeans. As in Sinorhizobium meliloti, a B. japonicum ndvA mutant was affected in salt-stress tolerance and exhibited symbiotic defects in that it induced the formation of ineffective soybean nodules. The central nodule tissue was infected by bacteroids, but within the infected cells the mutant was not properly maintained. Another gene was found to be highly similar to bacterial aspartases and thus was named aspA. The putative function of the product of this gene was confirmed by genetic complementation of aspartase-less Escherichia coli strain TK237. The symbiotic phenotype of a B. japonicum aspA:Tn KPK2 mutant consisted of enlarged symbiosomes that made the system ineffective. In general, Tn KPK2 is a suitable means for fast sequencing. In combination with pJQ200SK, the resulting recombinant plasmids can be directly used to create genetically defined mutants.

ATP-Binding Cassette Transporters↗
PubMed2004

Interaction of fibronectin type II proteins with membranes: the stallion seminal plasma protein SP-1/2.

Seminal plasma of mammalians contains, among others, proteins that are characterized by the fibronectin (Fn) type II module. Our knowledge about the structure and the physiological function of seminal Fn type II proteins mainly originates from studies on PDC-109, the bovine representative of this protein family. The present work focuses on the equine protein SP-1/2 (also named HSP-1/2) with particular emphasis on its interaction with lipid membranes by employing the intrinsic protein fluorescence and a number of spin-labeled and fluorescent lipid analogues. The results indicate that the interaction of SP-1/2 with (lipid) membranes is similar to that of PDC-109 which can be explained by homologous amino acid sequences of both proteins. Like PDC-109, SP-1/2 has a specificity for phospholipids with the phosphocholine headgroup. Upon binding to lipid vesicles, the protein intercalates into the hydrophobic membrane core, resulting in a rigidification of the lipid phase and, at higher concentration, in a perturbation of membrane structure. However, compared with PDC-109, the impact of SP-1/2 on membranes is less intense in that the degree of protein-mediated immobilization of lipids was lower. Furthermore, different to PDC-109, SP-1/2 was not able to extract lipids from human red blood cells. The data are discussed with regard to similarities and species-specific differences of the function of seminal Fn type II proteins in the genesis of sperm cells.

Amino Acid Motifs↗
PubMed2004

Basic leucine zipper transcription factors C/EBP and MafL in the hydrozoan jellyfish Podocoryne carnea.

Members of the CCAAT/enhancer binding protein (C/EBP) and the Maf protein subfamilies have been characterized in a variety of bilaterian organisms. This is the first report of C/EBP and MafL genes in a basal organism, the hydrozoan jellyfish Podocoryne carnea. Transcripts of both genes are present in all life cycle stages: egg, embryo, larva, polyp, and medusa. During early development, both factors appear to regulate metamorphosis of the larva to the primary polyp. Both genes are also expressed in the striated muscle of the developing and adult medusa. During in vitro transdifferentiation of striated muscle cells to smooth muscle and nerve cells, C/EBP is continuously expressed, whereas MafL expression is turned off during transdifferentiation and reactivated when nerve cells differentiate. Thus, both factors may be involved in muscle and nerve cell differentiation. In the mature medusa both genes are also implicated in gametogenesis. Developmental and evolutionary aspects of the gene structures and expression patterns are discussed.

Amino Acid Sequence↗
PubMed2004

Homologs of vascular endothelial growth factor and receptor, VEGF and VEGFR, in the jellyfish Podocoryne carnea.

Vascular endothelial growth factors (VEGF) are the major inducers of vasculogenesis and angiogenesis in vertebrates. Their effects are mediated by receptor tyrosine kinases of the VEGF receptor (VEGFR) family located on endothelial cells and include stimulation of cell survival, proliferation, migration, and tube formation as well as regulation of vascular permeability. Here, we report the presence of VEGF and VEGFR homologous genes in a basal invertebrate of the phylum Cnidaria. The marine jellyfish Podocoryne carnea features a gastrovascular system consisting of the feeding organ, or manubrium, the radial and ring canals, and the tentacle bulbs. Expression analysis indicates that both genes are involved in tentacle and gastrovascular canal formation, indicating an early recruitment of the VEGF signalling pathway for morphogenetic processes leading to tube formation in metazoans. The evolutionary origin of the VEGF signalling pathway resides in the common ancestor of the Cnidaria and Bilateria.

Amino Acid Sequence↗
PubMed2004

A novel genetic locus outside the symbiotic island is required for effective symbiosis of Bradyrhizobium japonicum with soybean Glycine max.

In order to investigate the symbiotic interaction between soybean and Bradyrhizobium japonicum, TnphoA mutagenesis of the microsymbiont was performed. Mutant strain 2-10 was found to induce a strongly reduced number of ineffective nodules. Ultrastructural analysis of the soybean nodule central tissue revealed the presence of numerous starch granules and vacuoles in the infected cells. In addition, the number of symbiosomes was extremely low, indicating an impaired interaction between the plant and invading bacteria. Cloning and sequencing of the mutated DNA region uncovered four open reading frames (ORFs) lacking any data base similarities. ORFs srrA1 and srrA2, the 2-10 TnphoA insertion site, are encoded in the same reading frame. A 35-kDa expression product in Escherichia coli indicated the presence of a common protein, called SrrA (symbiotically relevant region) in B. japonicum 110spc4, encoded by combined srrA1 and srrA2 genes. The analysis of gene disruption mutants revealed that srrB and srrC were also required for effective symbiosis with soybeans. Further downstream the gene for a putative inner membrane protein (pipA) of unknown function was encoded on the opposite strand. Primer extension studies led to the conclusion that the organization of genes differed from the RhizoBase annotation in this particular region of B. japonicum USDA110.

Amino Acid Sequence↗
PubMed2003

Aminophospholipids have no access to the luminal side of the biliary canaliculus: implications for thr specific lipid composition of the bile fluid.

About 95% of the bile phospholipids are phosphatidylcholine. Although the fractions of phosphatidylcholine and of both aminophospholipids phosphatidylserine and phosphatidylethanolamine in the canalicular membrane are in the same order of about 35% of total lipids, both aminophospholipids are almost absent from the bile. To rationalize this observation, we studied the intracellular uptake of various fluorescent phospholipid analogues and their subsequent enrichment in the bile canaliculus (BC) of HepG2 cells. Diacylaminophospholipid analogues but not phosphatidylcholine analogues became rapidly internalized by an aminophospholipid translocase (APLT) activity in the plasma membrane of HepG2 cells. We observed only low labeling of BC by diacylaminophospholipids but extensive staining by phosphatidylcholine analogues. In the presence of suramin, known to inhibit APLT, a strong labeling of BC by diacylaminophospholipid analogues was found that declined to a level observed for control cells after removal of suramin. Unlike diacylphosphatidylserine, diether phosphatidylserine analogue, which is not an appropriate substrate of APLT, accumulated in the BC. The correlation between low labeling of BC and an APLT-mediated transbilayer movement suggests the presence of an APLT activity in the canalicular membrane that prevents exposure of aminophospholipids to the bile.

Bile↗
PubMed2003

Signal transduction in the visual cascade involves specific lipid-protein interactions.

In retinal rod photoreceptor cells, transducin (Gt) and cyclic GMP phosphodiesterase (PDE) are peripherally anchored to the cytoplasmic surface of the disk saccules. We have examined the role of specific phospholipids in the interaction of these proteins with native osmotically intact disk vesicles, employing spin-labeled phospholipid analogues (2% of total phospholipids) and bovine serum albumin back-exchange assay. Inactive GDP-bound transducin exclusively reduced the extraction of negatively charged phosphatidylserine. The effect disappeared upon activation of the G-protein with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). PDE affected the extraction of the zwitterionic phosphatidylcholine and, to a smaller extent, of phosphatidylethanolamine. When active GtGTPgammaS interacted with the PDE to form the active effector, the interaction with phosphatidylcholine was specifically enhanced. Each copy of the G-protein bound 3 +/- 1 molecules of phosphatidylserine, whereas the PDE bound a much larger amount (70 +/- 10) of a mixture of phosphatidylcholine and ethanolamine. The results are interpreted as a head group-specific and state-dependent interaction of the signaling proteins with the phospholipids of the photoreceptor membrane.

Animals↗
PubMed2003

Evolutionary aspects of developmentally regulated helix-loop-helix transcription factors in striated muscle of jellyfish.

The function of basic helix-loop-helix (bHLH) proteins in cell differentiation was shown to be conserved from Drosophila to vertebrates, exemplified by the function of MyoD in striated muscle differentiation. In phylogeny striated muscle tissue appears first in jellyfish and the question of its evolutionary position is controversially discussed. For this reason we have studied the developmental role of myogenic bHLH genes in medusa development. Based on their dimerization ability, four genes of the bHLH family of transcription factors were isolated from the hydrozoan jellyfish Podocoryne carnea. While the proteins Id and Ash group with cognate family members from bilaterians, Net-like and JellyD1 could not be unequivocally classified. Id is expressed during the medusa budding process and in the adult medusa, Ash and Net-like are expressed in all life cycle stages from egg to adult medusa and JellyD1 is expressed in the blastula and gastrula stages, the planula larva, and in late medusa bud stages. The dimerization specificity, the expression pattern, and the conservation of two residues specific for a MyoD bHLH domain suggest that JellyD1 is related to an ancestral MyoD gene. Id, Net-like, and JellyD1 are either expressed in the entocodon or its derived tissues, the striated and smooth muscle of the bell. These findings strengthen the hypothesis that the entocodon is a mesoderm-like structure and that the common ancestor of Cnidaria and Bilateria was more complex in cell-type architecture and body organization than commonly thought.

Amino Acid Sequence↗