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  • SECONDARY STRUCTURE  (5)
  • BIOLOGY  (4)
Keywords
  • 1
    Keywords: RECEPTOR ; SPECTRA ; GENE ; PROTEINS ; BINDING ; BIOLOGY ; ELEMENTS ; STABILITY ; CD4-BINDING DOMAIN ; CONFORMATIONAL SWITCH ; GP120 ; FAMILIES ; POTENTIALS ; SET ; protein conformation ; CD-spectroscopy ; CHAIN INTERACTION INDEX ; conformational rigidity ; helix-inducing tetrads ; peptide folding ; switch peptides
    Abstract: In some naturally occurring protein sequences, an abrupt, concerted refolding from beta-sheet to helical conformation occurs when the polarity of the surrounding medium drops below a critical level. This switch-like behaviour was first observed on the HIV-1 envelope glycoprotein gp120, where it plays a crucial role in the efficient binding of gp120 to the T-cell receptor CD4. Previous work had shown that an N-terminal amino acid tetrad LPCR and a Trp located 5-20 residues downstream to the tetrad are common motifs in polarity-driven switch peptides. The LPCR tetrad governs the folding of the subsequent residues and acts as a helix initiation site, whereas the Trp is responsible for the cooperative character of the structural change due to multiple, simultaneous interactions of its quadrupole moment with several amino acid residues within the sequences. Here we identify and characterize new families of switch peptides that use different, turn-probable tetrads (LPST and VPSR) as helix initiation sites at the N-terminus. We have also been able to demonstrate that some tetrads with extremely high turn probability do not serve as helix initiation sites. Comparison of these with LPCR and the newly discovered tetrads LPST and VPSR has allowed a more comprehensive description of the physico-chemical properties of helix-inducing tetrads. The deeper understanding of the intrinsic properties of switch sequences allows the design of artificial polarity-driven switches, applicable in engineering of, e. g. controllable binding sites in artificial proteins.
    Type of Publication: Journal article published
    PubMed ID: 20878680
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  • 2
    Keywords: PEPTIDE ; CELLS ; GROWTH ; IN-VITRO ; tumor ; BLOOD ; CELL ; Germany ; IN-VIVO ; MODEL ; THERAPY ; VITRO ; VIVO ; imaging ; screening ; cell line ; ACCUMULATION ; LINES ; NUCLEAR-MEDICINE ; TIME ; SERA ; primary ; BINDING ; CELL-LINES ; SEQUENCE ; SEQUENCES ; ACID ; MOUSE ; ASSAY ; CELL-LINE ; LINE ; DISPLAY ; CELLULAR UPTAKE ; PEPTIDES ; BIODISTRIBUTION ; STABILITY ; HEAD ; NECK ; CLEARANCE ; MOUSE MODEL ; KINETICS ; specificity ; SECONDARY STRUCTURE ; cell lines ; nuclear medicine ; AFFINITY ; PHAGE DISPLAY ; SERUM ; targeting ; radiology ; RE ; INCREASE ; LIBRARIES ; LEVEL ; methods ; TUMOR-CELL ; ASSAYS ; INTERNALIZATION ; NUCLEAR ; MCF-7 CELLS ; technique ; pharmacology ; USA ; uptake ; tumor targeting ; in vivo ; MEDICINE ; TOO ; binding affinity ; MODIFIED PEPTIDE ; CONJUGATION ; BINDING PEPTIDE ; BLOOD-LEVELS ; circular dichroism ; peptide structure
    Abstract: The transfer of peptide sequences identified by screening of phage-displayed libraries to clinical application is often difficult. This study investigated whether coupling of a new peptide, FROP-1, to the chelator 1,4,7,10-tetraazacyclododecane-1,4, 7,10-tetraacetic acid (DOTA) resulted in structural restriction and, consequently, improved binding and stability. Methods: The peptide FROP-1 was coupled to the chelator DOTA and labeled with In-111. The structural changes caused by the addition of the chelator were determined by circular dichroism. The properties of this modified peptide were investigated in in vitro binding assays and monitored for kinetics, competition, and internalization as well as serum stability. A cell-type binding profile was established and the in vivo biodistribution was evaluated in a nude mouse model. Results: When compared with the free peptide without chelator, FROPDOTA revealed different cellular uptake kinetics, reaching a maximum at 2 h in vitro. The cells completely accumulated the tracer, and competition experiments revealed that 99.4% (FRO82-2 cells), 98.6% (MCF-7 cells), or 99.3% (average for 3 primary head and neck tumor cell lines) of tracer accumulation could be suppressed, revealing the specificity of this process. The internalization kinetics determined in MCF-7 cells supported this finding: After an incubation time of 180 min, the major fraction of FROPDOTA was trapped intracellularly. Serum stability experiments revealed an increase in stability due to the chelator, with a half-life of 71 min. Circular dichroism measurements indicated a fixed alpha-helix structure of FROPDOTA representing a strong change in secondary structure. In competition binding experiments, the binding constant (K-D) to FRO82-2 cells was determined to be 494 nM. Despite this avid binding affinity, the binding kinetics were found to be too slow to induce an uptake in vivo before clearance. Consequently, the biodistribution revealed a rapid renal and hepatobiliary clearance, with blood levels dropping from 5.48 +/- 0.26 %lD/g (percentage injected dose per gram) 5 min after injection to 0.77 +/- 0.15 %ID/g at 135 min after injection. Conclusion: This study revealed that peptides that are identified by display techniques may be underrated. Careful alteration of their structure will permit going beyond the possibilities that the limited pool of naturally occurring peptides provide for tumor targeting
    Type of Publication: Journal article published
    PubMed ID: 17704241
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  • 3
    Keywords: PEPTIDE ; SPECTRA ; Germany ; PROTEIN ; BIOLOGY ; MOLECULAR-BIOLOGY ; FUSION ; PEPTIDES ; SECONDARY STRUCTURE ; molecular biology ; molecular ; CHEMISTRY ; methods ; USA ; SPECTRUM
    Type of Publication: Journal article published
    PubMed ID: 17320030
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  • 4
    Keywords: ENVIRONMENT ; APOPTOSIS ; EXPRESSION ; GROWTH ; tumor ; CELL ; Germany ; human ; KINASE ; SITE ; TISSUE ; BINDING ; BIOLOGY ; fibroblasts ; MOLECULAR-BIOLOGY ; PHOSPHORYLATION ; ACID ; DESIGN ; MODULATION ; DERIVATIVES ; LIGANDS ; NETHERLANDS ; LECTIN ; STRUCTURAL-CHANGES ; HUMAN NEUROBLASTOMA-CELLS ; molecular biology ; molecular ; RE ; INCREASE ; NUCLEAR ; enzymatic ; modification ; FUNCTIONALITY ; amino acids ; MAMMALIAN LECTINS ; CARBOHYDRATE-BINDING PROTEIN-35 ; GANGLIOSIDE GM(1) ; histochemistry ; serine phosphorylation ; titania chromatography
    Abstract: Galectin-3 has a unique modular design. Its short N-terminal stretch can be phosphorylated, relevant for nuclear export and anti-anoikis/apoptosis activity. Enzymatic modification by casein kinase 1 at constant ATP concentration yielded mg quantities of mono- and diphosphorylated derivatives at Ser5/Ser11 in a 2:1 ratio. Their carbohydrate-inhibitable binding to asialofetuin, cell surfaces of three tumor lines, rabbit erythrocytes leading to haemagglutination and cytoplasmic sites in fixed tissue sections was not markedly altered relative to phosphate-free galectin-3. Spectroscopically, phosphorylation induced alterations in the far UV CD, indicative of an increase in ordered structure. This is accompanied by changes in the environment of aromatic amino acids signified by shifts in the near UV CD. (C) 2008 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 18302943
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  • 5
    Keywords: PEPTIDE ; IN-VITRO ; Germany ; MODEL ; GENERATION ; DISEASE ; antibodies ; antibody ; OLIGOMERS ; BETA ; PRECURSOR PROTEIN ; SECONDARY STRUCTURE ; DIMER ; FIBRILS ; SENILE PLAQUES
    Type of Publication: Journal article published
    PubMed ID: 12840025
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  • 6
    Keywords: PEPTIDE ; RECEPTOR ; Germany ; DISTINCT ; PROTEIN ; PROTEINS ; SEQUENCE ; SEQUENCES ; DESIGN ; MEMBRANE ; FUSION ; PEPTIDES ; SERIES ; AMINO-ACIDS ; SECONDARY STRUCTURE ; DE-NOVO ; RECEPTORS ; FLEXIBILITY ; MEMBRANES ; MEMBRANE PROTEIN ; MEMBRANE-PROTEIN ; SEGMENT ; RESIDUES ; ENVIRONMENTS ; GLYCINE ; INFLUENZA HEMAGGLUTININ ; INFRARED-SPECTROSCOPY ; PROPENSITIES ; SNARE PROTEINS ; SPANNING DOMAIN ; VIRUS G-PROTEIN
    Abstract: Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of alpha-helix-promoting Leu and beta-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an alpha-helical state to a beta-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu/ Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity
    Type of Publication: Journal article published
    PubMed ID: 15456911
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  • 7
    Keywords: PEPTIDE ; Germany ; PROTEIN ; PROTEINS ; MECHANISM ; BIOLOGY ; MOLECULAR-BIOLOGY ; MEMBRANE ; FUSION ; FUSION PROTEINS ; CRYSTAL-STRUCTURE ; PEPTIDES ; SECONDARY STRUCTURE ; DOMAINS ; FUSION PROTEIN ; molecular biology ; molecular ; RE ; GLYCINE ; INFLUENZA HEMAGGLUTININ ; MUTATIONAL ANALYSIS ; VIRAL MEMBRANE-FUSION ; analysis ; ENVELOPE PROTEIN ; TRANSMEMBRANE DOMAIN ; viral ; circular dichroism ; AVIAN-SARCOMA ; LEUKOSIS VIRUS ; membrane fusion ; VIRUS GLYCOPROTEIN-G
    Abstract: Membrane fusion is essential for many biological processes. Though there hove been many structure and fusion studies of cel lular and viral fusion proteins in the last years, their functional mechanism remains elusive. In particular, the structural modes of operation of the transmembrane domains and viral fusion peptides of fusion proteins during membrane fusion have not been elucidated, although work on de novo designed fusogenic peptides suggested that conformational flexibility was necessary. In addition, the use of different and incompatible measurement criteria has made a comparative overview difficult. Here, we report a systematic structural analysis of viral fusion peptides from different fusion protein classes and transmembrane domains of viral and cellular fusion proteins by using circular dichroism spectroscopy. The data that were obtained demonstrate that class viral fusion peptides show a structural flexibility between helix and irregular secondary structures, whereas fusion peptides of class II viral fusion proteins are characterized by a stable random coil and turn structure. Thus, conformational flexibility does not seem to be a universal criterion for the fusion activity of a fusion peptide. On the contrary, the transmembrane domains of fusion proteins are distinguished by a structural flexibility between helix and sheet structure that is similar to de novo designed unnatural peptides with high fusion activities (M. W. Hofmann et al. PNAS 2004, 101, 14776-14781). Thus, the conformational behavior of the fusogenic unnatural peptides most closely resembles that of, fusion protein transmembrane domains, and allows them to be used to gain a deeper understanding of the membrane fusion process
    Type of Publication: Journal article published
    PubMed ID: 18330852
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