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  • 1
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    German Medical Science GMS Publishing House; Düsseldorf
    In:  127. Kongress der Deutschen Gesellschaft für Chirurgie; 20100420-20100423; Berlin; DOC10dgch219 /20100517/
    Publication Date: 2010-05-17
    Keywords: ddc: 610
    Type: conferenceObject
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  • 2
    Keywords: PEPTIDE ; SIMULATIONS ; SPECTRA ; Germany ; PROTEINS ; DYNAMICS ; SIMULATION ; PARAMETERS ; VACCINE ; KINETICS ; ALPHA-HELIX ; CD4-BINDING DOMAIN ; CONFORMATIONAL SWITCH ; EXPLICIT SOLVENT WATER ; GP120 ; IMMUNOGENIC PEPTIDE ; molecular dynamics simulation,peptide,convergence ; MOLECULAR-DYNAMICS SIMULATION ; TRIFLUOROETHANOL
    Abstract: To examine the conformational properties in aqueous solution of a 15-residue peptide that is a potential pharmacophore for AIDS vaccine development, molecular dynamics simulations were performed in water starting from structures determined experimentally in three different organic solvents. Convergence characteristics of the simulation are examined in Cartesian and conformational spaces. In addition, novel analysis tools are employed including a multidimensional scaling method to represent the distance between trajectory frames. As these methods are based on a variety of physical parameters, they provide a useful cross-check on the structural convergence. Theoretical two-dimensional (2D) H-1-NMR spectra are also generated. These are superficially quite different in appearance, demonstrating that backbone similarities difficult to identify by visual inspection of 2D NMR data can be revealed using the methods described here. (C) 2003 Wiley Periodicals, Inc
    Type of Publication: Journal article published
    PubMed ID: 14517902
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  • 3
    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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  • 4
    Keywords: PEPTIDE ; Germany ; COMMON ; PROTEIN ; PROTEINS ; DOMAIN ; ANTIGEN ; DYNAMICS ; SIMULATION ; SEQUENCE ; SEQUENCES ; antibodies ; antibody ; TARGET ; DESIGN ; VARIABILITY ; VACCINE ; STRATEGIES ; PREDICTION ; GP120 ; MOLECULAR-DYNAMICS ; HIV-1 ; GENOMIC DIVERSITY ; HTLV-III ; MINIMIZATION
    Abstract: The most promising target antigen for an HIV vaccine designed using the classic antibody strategy has been the viral coat protein gp120. Unfortunately, its high variability has prevented this approach. We examine here a 15-residue peptide derived from the CD4-binding domain of gp120. By use of molecular dynamics computer simulation, it is shown that despite considerable sequence variation, the three-dimensional structure of the peptide is preserved over the full range of clade-specific sequences. Furthermore, sequences threaded onto the structure exhibit common three-dimensional electrostatic and hydrophobic properties. These common physicochemical characteristics constitute a pharmacophoric footprint that promises to be useful in the design of a synthetic antigen for vaccine development
    Type of Publication: Journal article published
    PubMed ID: 15239651
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  • 5
    Abstract: ABSTRACT The delivery of molecules into cells poses a critical problem that has to be solved for the development of diagnostic tools and therapeutic agents acting on intracellular targets. Cargos which by themselves cannot penetrate cellular membranes due to their biophysical properties can achieve cell membrane permeability by fusion to protein transduction domains (PTDs). Here, we engineered a universal delivery system based on PTD-fused Strep-Tactin (ST) which we named Transtactin. Biochemical characterization of Transtactin variants bearing different PTDs indicated high thermal stabilities and robust secondary structures. Internalization studies demonstrated that Transtactins facilitated simple and safe transport of Strep-tag II-linked small molecules, peptides, and multicomponent complexes, or biotinylated proteins into cultured human cells. Transtactin-introduced cargos were functionally active, as shown for horseradish peroxidase serving as a model protein. Our results demonstrate that Transtactin provides a universal and efficient delivery system for Strep-tag II-fused cargos.
    Type of Publication: Journal article published
    PubMed ID: 19602053
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  • 6
    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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  • 7
    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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  • 8
    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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  • 9
    Keywords: ENERGIES ; PEPTIDE ; Germany ; LINES ; DOMAIN ; IONS ; BINDING ; SEQUENCE ; ACID ; ACIDS ; ENERGY ; BEHAVIOR ; CONFORMATIONAL SWITCH ; GP120 ; HIV-1 ; CHAIN ; RE ; interaction ; HIV ; SWITCH ; USA ; POLARITY ; GAUSSIAN-BASIS SETS ; MEDIA ; ATOMS LI ; interactions ; amino acids ; POSITION ; APPROXIMATE COULOMB POTENTIALS ; AUXILIARY BASIS-SETS ; KR ; PI INTERACTIONS
    Abstract: A 15-residue sequence (LPCRIKQFINMWQEV) forming the principal CD4-binding domain of gp120 from HIV 1 displays unusual, highly cooperative refolding from beta-hairpin to 3(10) helix when the polarity of the surrounding medium drops below a critical point, the so-called conformational switch. The tryptophan at position 12 has been shown to be essential for the cooperativity of the refolding process, and several lines of evidence from earlier work had suggested that it was the aromatic quadrupole that was responsible for this. To de. ne more precisely what physico-chemical properties of tryptophan brought about the unique behavior of this peptide, nonproteogenic aromatic amino acids have been selected based on desired alterations in quadrupole moment, electrostatic potential surface, and binding energy to ions. These were built into the peptide in the place of tryptophan and their effect on switch behavior examined. It could be shown that a minimal strength of the quadrupole moment is necessary but not sufficient to enforce cooperativity of refolding, with other properties of tryptophan playing a role in the optimum interaction of this residue with other side chains of the peptide
    Type of Publication: Journal article published
    PubMed ID: 18599633
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  • 10
    Keywords: FACTOR RECEPTOR ; ACTIVATION ; DOMAIN ; BINDING ; BOVINE PAPILLOMAVIRUS ; TRANSFORMATION ; EPITHELIAL-CELL LINE ; ONCOPROTEIN ; GOLGI ; CIRCULAR-DICHROISM
    Abstract: The product of the E5 oncogene in human papillomaviruses (HPVs) participates in cellular transformation. The sequences of E5 from high-risk HPV types are closely related, and the ability to transform is thought to be associated with their structure. Structural determination by standard biophysical methods has proved impossible due to the extreme hydrophobicity of the gene product. We have achieved limited solubility by dividing the sequence into three, structurally distinct domains. Synthetic peptides corresponding to these domains have been examined using circular dichroism (CD) spectroscopy, a method that can detect secondary structure elements in highly dilute protein solutions. Using data on the secondary structure content of these domains under different conditions and in systematic combination to detect constructive domain interactions, a model of HPV E5 structure and position in the membrane is proposed that is consistent with what is known of the larger family of leucine-rich repeat (LRR) proteins to which it belongs.
    Type of Publication: Journal article published
    PubMed ID: 12429498
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