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  • 1
    Keywords: PEPTIDE ; Germany ; COMMON ; INFORMATION ; TOOL ; SITE ; PROTEIN ; PROTEINS ; INDEX ; BINDING ; SEQUENCE ; SEQUENCES ; SIGNAL ; ACID ; GLYCOPROTEIN ; DESIGN ; PEPTIDES ; PARAMETERS ; STABILITY ; BEHAVIOR ; INITIATION ; GP120 ; CHAIN ; PROTEIN DESIGN
    Abstract: Certain sequences within proteins have the ability to undergo an abrupt cooperative conformational switch from beta-strand to helix in response to decreasing polarity of the environment. This behavior was first observed at the CD4 binding site of the envelope glycoprotein gp120 of HIV-1, but evidence has accumulated that polarity-driven beta --〉 alpha switches may be widespread, serving both to facilitate binding on protein/membrane or protein/protein contact and to signal that docking has occurred. The characteristics identified so far that distinguish switch sequences (a reverse turn at the N-terminus that acts as a helix initiation site, a conserved tryptophan residue downstream, and high potential for both the helix and beta-fold) appear to be necessary but not sufficient, as some otherwise promising sequences found in data bank searches proved not to be capable of cooperative refolding. Analysis of existing switches has led to the development of the side chain interaction index (SCII) as a further parameter characterizing the beta --〉 alpha polarity-driven switch. Data bank searches using this additional parameter have successfully identified a series of new potential switch sequences. All of them have in common the amino acid tetrad LPCR at the N-terminus and a tryptophan 5-20 residues C-terminal to it. Those with a high SCII as well, when synthesized and tested, exhibited strongly cooperative polarity-driven refolding. Control peptides, containing all other parameters but with a low SCII, did not. Using this new information, an artificial sequence was designed that had a high SCII as well as the initiation site, conserved tryptophan, and high P-alpha and P-beta. When synthesized and tested, this sequence did in fact behave as a conformational switch, refolding cooperatively from beta-fold to helix at a threshold value of 30% TFE. The successful design of a polarity-driven conformational switch opens the possibility of using this motif as a tool in protein engineering
    Type of Publication: Journal article published
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  • 2
    Abstract: The transcription activator protein NtrC (nitrogen regulatory protein C, also termed NR(I)) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma(54) factor (RNAP x sigma(54)) from the closed complex (RNAP x sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC (NtrC-P), assembly of an octameric NtrC-P complex at the enhancer DNA sequence, interaction of this complex with promoter-bound RNAP x sigma(54) via DNA looping, and hydrolysis of ATP. Here it is demonstrated by two-color fluorescence cross-correlation spectroscopy measurements of 6-carboxyfluorescein and 6-carboxy-X-rhodamine-labeled DNA oligonucleotide duplexes that the NtrC-P complex can bind two DNA duplexes simultaneously. This suggests a model for the conformation of the looped intermediate that is formed between NtrC-P and RNAP. sigma(54) at the glnAp2 promoter during the activation process.
    Type of Publication: Journal article published
    PubMed ID: 10694378
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  • 3
    Keywords: Germany ; DNA ; SIGNAL ; ENERGY ; acetylation ; FLUORESCENCE ; ANGSTROM RESOLUTION ; CRYOELECTRON MICROSCOPY ; NUCLEOSOME CORE PARTICLE ; higher-order structure ; SUPERCOILED DNA ; NUCLEOSOME ; HISTONE ACETYLATION ; histone H1 ; CHROMATIN FIBER ; CONFORMATION ; LIQUID ; SCANNING FORCE MICROSCOPY ; DISTANCES ; H1 ; LINKER HISTONE H1 ; NEUTRON-SCATTERING
    Abstract: The effect of the salt concentration, linker histone H1, and histone acetylation on the structure of trinucleosomes reconstituted on a 608 bp DNA containing one centered nucleosome positioning signal was studied. Fluorescence resonance energy transfer (FRET) in solution and scanning force microscopy (SFM) measurements in liquid were done on the same samples. The distance between the DNA ends decreases under the effect of an increasing salt concentration and also by the incorporation of the H1 linker histone. A decrease of internucleosomal center-to-center (cc) distances by H1 was observed that was limited to a minimal value of about 20 nm. The distribution of the angle formed between consecutive nucleosomes was narrowed by H1. The effect of acetylation of all histones leads to decompaction, measured as an increased distance between the DNA ends, and also increased the internucleosomal distances. Selective acetylation of histone H4, however, compacts the structure as measured by FRET
    Type of Publication: Journal article published
    PubMed ID: 16953569
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  • 4
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    Biochemistry 45 (6), 1591-1598 
    Keywords: CELLS ; Germany ; IN-VIVO ; SITE ; DNA ; DOMAIN ; STABILITY ; NUCLEOSOME CORE PARTICLE ; RE ; higher-order structure ; HELA-CELLS ; NUCLEOSOME ; analytical ultracentrifugation ; LEVEL ; HISTONE ACETYLATION ; LINKER DNA ; H1 ; NEUTRON-SCATTERING ; RECONSTITUTION ; TAILS
    Abstract: Using a previously described FRET technique, we measured the distance between the ends of DNA fragments on which nucleosomes were reconstituted from recombinant and native histories. This distance was analyzed in its dependence on the DNA fragment length, concentration of mono- and divalent counterions, presence of linker historic H1, and historic modifications. We found that the linker DNA arms do not cross under all conditions studied but diverge slightly as they leave the histone core surface. Historic H I leads to a global approach of the linker DNA arms, confirming the notion of a "stem structure". Increasing salt concentration also leads to an approach of the linker DNAs. To study the effect of acetylation, we compared chemically acetylated recombinant histories with histories prepared from HeLa cells, characterizing the sites of acetylation by mass spectroscopy. Nucleosomes from chemically acetylated histories have few modifications in the core domain and form nucleosomes normally. Acetylating all histories or selectively only H3 causes an opening of the nucleosome structure, indicated by the larger distances between the linker DNA ends. Selective acetylation of H4 distances the linker ends for short fragments but causes them to approach each other for fragments longer than 180 bp
    Type of Publication: Journal article published
    PubMed ID: 16460006
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  • 5
    Keywords: RECEPTOR ; CELL ; Germany ; MODEL ; DENSITY ; VOLUME ; GENE ; TRANSDUCTION ; ACTIVATION ; COMPLEX ; RESPONSES ; COMPLEXES ; DOMAIN ; BIOLOGY ; MOLECULAR-BIOLOGY ; signal transduction ; SIGNAL ; MUTANT ; AMPLIFICATION ; SIGNAL-TRANSDUCTION ; EFFICIENT ; LOCALIZATION ; PROGENITOR CELLS ; CELL-SURFACE ; point mutation ; DIMERIZATION ; DOMAINS ; ERYTHROPOIETIN ; ERYTHROPOIETIN RECEPTOR ; LAYER ; OLIGOMERIZATION ; signaling ; molecular biology ; molecular ; RE ; VARIANT ; RESPONSIVENESS ; analysis ; MUTANTS ; TRANSMEMBRANE DOMAIN ; USA ; correlation ; SET ; NOV ; correlates ; modeling ; response ; erythrocytosis ; FUNCTIONALITY ; biological ; MEMBRANE-PROTEINS ; PRIMARY FAMILIAL POLYCYTHEMIA
    Abstract: The formation of signal-promoting dimeric or oligomeric receptor complexes at the cell surface is modulated by self-interaction of their transmembrane (TM) domains. To address the importance of TM domain packing density for receptor functionality, we examined a set of asparagine mutants in the TM domain of the erythropoietin receptor (EpoR). We identified EpoR-T242N as a receptor variant that is present at the cell surface similar to wild-type EpoR but lacks visible localization in vesicle-like structures and is impaired in efficient activation of specific signaling cascades. Analysis by a molecular modeling approach indicated an increased interhelical distance for the EpoR-T242N TM dimer. By employing the model, we designed additional mutants with increased or decreased packing volume and confirmed a correlation between packing volume and biological responsiveness. These results propose that the packing density of the TM domain provides a novel layer for fine-tuned regulation of signal transduction and cellular decisions
    Type of Publication: Journal article published
    PubMed ID: 18855427
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  • 6
    Keywords: INHIBITION ; PROTEIN ; ACTIVATION ; MECHANISM ; BINDING ; SUBUNIT ; MODULATION ; CALCIUM ; ion channels ; STOICHIOMETRY ; olfactory receptor neurons ; CA2+-DEPENDENT INTERACTIONS ; CNG CHANNELS ; ODOR ADAPTATION ; TERMINAL REGIONS
    Abstract: Cyclic nucleotide-gated (CNG) channels operate as transduction channels in photoreceptors and olfactory receptor neurons. Direct binding of cGMP or cAMP opens these channels which conduct a mixture of monovalent cations and Ca2+. Upon activation, CNG channels generate intracellular Ca2+ signals that play pivotal roles in the transduction cascades of the visual and olfactory systems. Channel activity is controlled by negative feedback mechanisms that involve Ca2+-calmodulin, for which all CNG channels possess binding sites. Here we compare the binding properties of the two LQ-type calmodulin binding sites, both of which are thought to be involved in channel regulation. They reside on the isoforms CNGB1 and CNGA4. The CNGB1 subunit is present in rod photoreceptors and olfactory receptor neurons. The CNGA4 subunit is only expressed in olfactory receptor neurons, and there are conflicting results as to its role in calmodulin-mediated feedback inhibition. We examined the interaction of Ca2+-calmodulin with two recombinant proteins that encompass either of the two LQ sites. Comparing binding properties, we found that the LQ site of CNGB1 binds Ca2+-calmodulin at 10-fold lower Ca2+ levels than the LQ site of CNGA4. Our data provide biochemical evidence against a contribution of CNGA4 to feedback inhibition. In accordance with previous work on photoreceptor CNG channels, our results indicate that feedback control is the exclusive role of the B-subunits in photoreceptors and olfactory receptor neurons
    Type of Publication: Journal article published
    PubMed ID: 21413724
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  • 7
    Keywords: ESCHERICHIA-COLI ; CRYSTAL-STRUCTURE ; PEPTIDE INHIBITOR ; NUCLEAR-MAGNETIC-RESONANCE ; ALKALINE-PHOSPHATASE ; RECOMBINANT CATALYTIC SUBUNIT ; BOVINE CARDIAC-MUSCLE ; PHOSPHOSERINE RESIDUES ; CLOSED CONFORMATIONS ; TROPONIN-I
    Abstract: Cell signaling pathways rely on phosphotransfer reactions that are catalyzed by protein kinases. The protein kinases themselves are typically regulated by phosphorylation and concurrent structural rearrangements, both near the catalytic site and elsewhere. Thus, physiological function requires posttranslational modification and deformable structures. A prototypical example is provided by cyclic AMP-dependent protein kinase (PKA). It is activated by phosphorylation, is inhomogeneously phosphorylated when expressed in bacteria, and exhibits a wide range of dynamic properties. Here we use (31)P nuclear magnetic resonance (NMR) spectroscopy to characterize the phosphorylation states and to estimate the flexibility of the phosphorylation sites of 2-, 3-, and 4-fold phosphorylated PKA. The phosphorylation sites Ser10, Ser139, Thr197, and Ser338 are assigned to individual NMR resonances, assisted by complexation with AMP-PNP and dephosphorylation with alkaline phosphatase. Rotational diffusion correlation times estimated from resonance line widths show progressively increasing flexibilities for phosphothreonine 197, phosphoserines 139 and 338, and disorder at phosphoserine 10, consistent with crystal structures of PKA. However, because the apparent rotational diffusion correlation time fitted for phosphothreonine 197 of the activation loop is longer than the overall PKA rotational diffusion time, microsecond to millisecond time scale conformational exchange effects involving motions of phosphothreonine 197 are probable. These may represent "open"-"closed" transitions of the uncomplexed protein in solution. These data represent direct measurements of flexibilities also associated with functional properties, such as ATP binding and membrane association, and illustrate the applicability of (31)P NMR for functional and dynamic characterization of protein kinase phosphorylation sites.
    Type of Publication: Journal article published
    PubMed ID: 11993991
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  • 8
    Keywords: ENERGIES ; measurement ; GROWTH ; CELL ; Germany ; human ; MODEL ; SITE ; PROTEIN ; DRUG ; COMPLEX ; LIGAND ; COMPLEXES ; BINDING ; LINKAGE ; SIGNAL ; PROTON ; SPECTROSCOPY ; MALIGNANCIES ; DESIGN ; ENERGY ; INTERACTION ENERGIES ; NMR ; SURFACE ; LIGANDS ; STRATEGIES ; SIALIC-ACID ; SELECTION ; CELL-SURFACE ; carbohydrates ; BUILDING-BLOCKS ; AFFINITY-CHROMATOGRAPHY ; ENDOGENOUS LECTINS ; HUMAN NEUROBLASTOMA-CELLS ; neuroblastoma ; CARBOHYDRATE-BINDING ; CHOLERA-TOXIN ; HEAT-LABILE ENTEROTOXIN ; LIGAND-BINDING ; MISTLETOE LECTIN ; MOLECULAR-DYNAMICS SIMULATIONS ; OLIGOSACCHARIDE CHAIN
    Abstract: Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM, with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and psi value of -70degrees and 15degrees vs 70degrees and 15degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin ((D and IF values of -172degrees and -26degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences
    Type of Publication: Journal article published
    PubMed ID: 14674750
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  • 9
    Keywords: Germany ; IN-VIVO ; ENZYMES ; PROTEINS ; TIME ; IONS ; CONTRAST ; BINDING ; BIOLOGY ; MOLECULAR-BIOLOGY ; VARIANTS ; ACID ; CLEAVAGE ; FORM ; DIFFERENCE ; REQUIRES ; CRYSTAL-STRUCTURE ; DIMER ; AFFINITY ; SINGLE ; molecular biology ; molecular ; VARIANT ; AMINO-ACID ; SUBSTRATE ; ENZYME ; LONG ; USA ; HIGH-AFFINITY ; BETA-LACTAMASE DOMAIN ; CANCER SUSCEPTIBILITY GENE ; ENDORIBONUCLEASE ; TRANSFER-RNA ; Z-FAMILY ; ZINC PHOSPHODIESTERASE
    Abstract: The endonuclease tRNase Z from A. thaliana (AthTRZ1) was originally isolated for its tRNA 3' processing activity. Here we show that AthTRZ1 also hydrolyzes the phosphodiester bond in bis(pnitrophenyl) phosphate (bpNPP) with a k(cat) of 7.4 s(-1) and a K-M of 8.5 mM. We analyzed 22 variants of AthTRZ1 with respect to their ability to hydrolyze bpNPP. This mutational mapping identified fourteen variants that lost the ability to hydrolyze bpNPP and seven variants with reduced activity. Surprisingly, a single amino acid change (R252G) resulted in a ten times higher activity compared to the wild type enzyme. tRNase Z enzymes exist in long and short forms. We show here that in contrast to the short tRNase Z enzyme AthTRZ1, the long tRNase Z enzymes do not have bpNPP hydrolysis activity pointing to fundamental differences in substrate cleavage between the two enzyme forms. Furthermore, we determined the metal content of AthTRZ1 and analyzed the metal requirement for bpNPP hydrolysis. AthTRZ1 shows a high affinity for Zn2+ ions; even upon incubation with metal chelators, 0.76 Zn2+ ions are retained per dimer. In contrast to bpNPP hydrolysis, pre-tRNA processing requires additional metal ions, Mn2+ or Mg2+, Zn2+ ions alone are insufficient
    Type of Publication: Journal article published
    PubMed ID: 18052196
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  • 10
    Keywords: IN-VITRO ; PROTEIN ; BINDING ; SPECTROSCOPY ; ESCHERICHIA-COLI ; FLUORESCENCE ; CONFORMATION ; STATE ; ENERGY CONTENT ; ALPHA-SYNUCLEIN ; GAINS STRUCTURE ; INTRINSICALLY UNSTRUCTURED PROTEINS ; TRIMETHYLAMINE-N-OXIDE
    Abstract: Crowding caused by the high concentrations of macromolecules in the living cell changes chemical equilibria, thus promoting aggregation and folding reactions of proteins. The possible magnitude of this effect is particularly important with respect to the physiological structure of intrinsically disordered proteins (IDPs), which are devoid of well-defined three-dimensional structures in vitro. To probe this effect, we have studied the structural state of three IDPs, alpha-casein, MAP2c, and p21(Cip1), in the presence of the crowding agents Dextran and Ficoll 70 at concentrations up to 40%, and also the small-molecule osmolyte, trimethylamine N-oxide (TMAO), at concentrations up to 3.6 M. The structures of IDPs under highly diluted and crowded conditions were compared by a variety of techniques, fluorescence spectroscopy, acrylamide quenching, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, fluorescence correlation spectroscopy (FCS), and far-UV and near-UV circular dichroism (CD) spectroscopy, which allow us to visualize various levels of structural organization within these proteins. We observed that crowding causes limited structural changes, which seem to reflect the functional requirements of these 1DPs. alpha-Casein, a protein of nutrient function in milk, changes least under crowded conditions. On the other hand, MAP2c and, to a lesser degree, p21(Cip1), which carry out their functions by partner binding and accompanying partially induced folding, show signs of local structuring and also some global compaction upon crowded conditions, in particular in the presence of TMAO. The observations are compatible with the possible preformation of binding-competent conformations in these proteins. The magnitude of these changes, however, is far from that of the cooperative folding transitions elicited by crowding in denatured globular proteins; i.e., these IDPs do remain in a state of rapidly interconverting structural ensemble. Altogether, our results underline that structural disorder is the physiological state of these proteins
    Type of Publication: Journal article published
    PubMed ID: 21634433
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