Supramolekulare Chemie mit einzelnen Molekülen

Eckel, Rainer; Ros, Robert; Decker, Björn; Mattay, Jochen; Anselmetti, Dario

doi:10.1002/ange.200461382

Cited by 9 publications

(3 citation statements)

References 33 publications

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“…A broad affinity range for specific molecular recognition between single binding partners from 10 À5 m [15] to 10 À15 m [16,17] can be investigated with single-molecule force spectroscopy. The deflection of a micro-fabricated force sensor (cantilever) is used to detect forces in the pico-Newton range.…”

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“…The AFM principle permits investigation of molecular binding forces under physiological conditions, and AFM force spectroscopy has found applications in the study of a variety of specific biological receptor-ligand interactions [16][17][18][19][20][21][22] as well as in supramolecular host-guest systems. [15] The direct investigation of specific, native protein-DNA interactions has been reported recently. [23][24][25] In our studies reported herein, we applied AFM singlemolecule force spectroscopy to study the specific binding between the peptides listed in Table 1 and the DNA target sequence.…”

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Single‐Molecule Experiments in Synthetic Biology: An Approach to the Affinity Ranking of DNA‐Binding Peptides

et al. 2005

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Gene expression in eukaryotes is controlled at the transcriptional level by the specific binding of transcription factors to defined DNA sequences. In this way, cell growth, differentiation, and development are regulated. The possibility to influence and control cell metabolism through modified synthetic transcription factors [1][2][3][4] offers fascinating prospects for molecular cell biology in the framework of biomimetics and synthetic biology. [5,6] The design and synthesis of biologically active artificial enzymes and new protein-based materials can be investigated by the combination of bioorganic bottom-up synthesis and single-molecule affinity nanotechnology. With this approach important questions can be addressed, such as the extent to which a single recognition helix contributes to the specific binding of a complete protein to DNA, the effect that a single amino acid point mutation has upon biological specificity and affinity, and the minimal peptide sequence length to ensure binding specificity. This approach would also aid in the design of artificial proteins that contain a purely synthetic helix-turn-helix (HTH) binding motif.In this context, it is of considerable interest to elucidate the DNA-binding specificity of synthetic peptides with a primary sequence akin to the binding domain of a transcription factor. We studied a 20-residue peptide that represents the native sequence of a binding epitope of the transcription activator PhoB (E. coli) and three single point mutants of this peptide.PhoB is a transcription activator which, after phosphorylation by PhoR, binds to the phosphate box in the promoter region of the phosphate regulon pho and activates the expression of genes involved in phosphate metabolism.

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Single‐Molecule Experiments in Synthetic Biology: An Approach to the Affinity Ranking of DNA‐Binding Peptides

et al. 2005

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“…A polymer linker, which is used to bind the PhoB‐DNA to the cantilever, ensures that the binding/dissociation event occurs far enough from the surface and that the molecular binding partners have enough steric flexibility to arrange themselves in an appropriate spatial manner. Furthermore, the typical nonlinear stretching of the polymer chain, which precedes the molecular dissociation event and reflects the elasticity of the polymer chain, facilitates the detection and discrimination of a specific dissociation event from unspecific binding 12, 26, 27. Unspecific tip–surface interactions occur closer to the surface and can therefore be discriminated.…”

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Single‐Molecule Experiments to Elucidate the Minimal Requirement for DNA Recognition by Transcription Factor Epitopes

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Körnig

et al. 2009

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Interactions between proteins and DNA are essential for the regulation of cellular processes in all living organisms. In this context, it is of special interest to investigate the sequence-specific molecular recognition between transcription factors and their cognate DNA sequences. As a model system, peptide and protein epitopes of the DNA-binding domain (DBD) of the transcription factor PhoB from Escherichia coli are analyzed with respect to DNA binding at the single-molecule level. Peptides representing the amphiphilic recognition helix of the PhoB DBD (amino acids 190-209) are chemically synthesized and C-terminally modified with a linker for atomic force microscopy-dynamic force spectroscopy experiments (AFM-DFS). For comparison, the entire PhoB DBD is overexpressed in E. coli and purified using an intein-mediated protein purification method. To facilitate immobilization for AFM-DFS experiments, an additional cysteine residue is ligated to the protein. Quantitative AFM-DFS analysis proves the specificity of the interaction and yields force-related properties and kinetic data, such as thermal dissociation rate constants. An alanine scan for strategic residues in both peptide and protein sequences is performed to reveal the contributions of single amino acid residues to the molecular-recognition process. Additionally, DNA binding is substantiated by electrophoretic mobility-shift experiments. Structural differences of the peptides, proteins, and DNA upon complex formation are analyzed by circular dichroism spectroscopy. This combination of techniques eventually provides a concise picture of the contribution of epitopes or single amino acids in PhoB to DNA binding.

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Einzelmolekülexperimente in der synthetischen Biologie – ein Ansatz für das Affinitätsranking DNA‐bindender Peptide

et al. 2005

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Peptid‐DNA‐Erkennung: Die spezifische Bindung von Peptiden, die einen Ausschnitt aus der Bindesequenz eines Transkriptionsaktivators repräsentieren, an DNA wurde erstmals auf Einzelmolekülniveau durch Kraftspektroskopie nachgewiesen (Versuchsprinzip siehe Bild). Die Ergebnisse unterstreichen die Bedeutung der Peptidlänge und kooperativer Effekte sowie den Beitrag einzelner Punktmutationen zur spezifischen Bindung.

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Supramolekulare Chemie mit einzelnen Molekülen

Cited by 9 publications

References 33 publications

Single‐Molecule Experiments in Synthetic Biology: An Approach to the Affinity Ranking of DNA‐Binding Peptides

Single‐Molecule Experiments in Synthetic Biology: An Approach to the Affinity Ranking of DNA‐Binding Peptides

Single‐Molecule Experiments to Elucidate the Minimal Requirement for DNA Recognition by Transcription Factor Epitopes

Einzelmolekülexperimente in der synthetischen Biologie – ein Ansatz für das Affinitätsranking DNA‐bindender Peptide

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