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

Eckel, Rainer; Wilking, Sven David; Becker, Anke; Sewald, Norbert; Ros, Robert; Anselmetti, Dario

doi:10.1002/ange.200500152

Cited by 3 publications

(9 citation statements)

References 34 publications

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“…Therefore, only the protein PhoB(127–229) ( 5 ) instead of PhoB(126–229) was obtained (see Table 1). In addition to the wild‐type protein ( 5 ), three protein point mutants ( 6 – 8 ) were accessible analogously to the previously analyzed peptides 12…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Resultsmentioning

confidence: 99%

“…E) Control experiment with EBNA‐DNA ( 13 ) containing TGTCA motifs without flanking regions important for DNA binding of PhoB and the PhoB wild‐type protein ( 5 ). F) Force spectroscopy experiment of wild‐type peptide ( 1 ) and Pho box DNA ( 11 )12. All experiments were measured in standard reaction buffer (100 m M NaH 2 PO 4 , 50 m M NaCl, pH 7.4) at 1000 nm s −1 retract velocity.…”

Section: Resultsmentioning

confidence: 99%

“…As previously communicated,12 the peptides derived from PhoB helix α 3 (PhoB(190–209); 1 ) and its point mutants ( 2 – 4 ) were investigated by using AFM–DFS measurements. Binding events were detected for the wild‐type peptide ( 1 ), and the peptide mutants R193A ( 2 ) and H198A ( 3 ).…”

Section: Resultsmentioning

confidence: 99%

“…In the class of zinc finger proteins, this is accomplished by attaching a minor groove tripyrrole binder to 31 amino acids representing the minimal DNA‐binding domain (DBD) of the GAGA factor of Drosophila melanogaster 8. In our studies, the transcription factor PhoB from Escherichia coli 9–11 was used as a model protein and the binding properties of its entire DBD were compared with those of synthetic 20‐mer peptides 12…”

Section: Introductionmentioning

confidence: 99%

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

Wollschläger

Gaus

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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Wollschläger

Gaus

Körnig

et al. 2009

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Fluorescence‐Emission Control of Single CdSe Nanocrystals Using Gold‐Modified AFM Tips

Eckel¹,

Walhorn²,

Pelargus³

et al. 2006

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A new means of switching the fluorescence emission of a single nanocrystal by intervention of a gold‐modified AFM tip, and the luminescence properties (see TIRFM image) as a function of distance between the single quantum dot and the tip, are presented. Significant luminescence enhancement due to exciton–plasmon coupling was detected. This is important in controlling molecular individuals with applications ranging from microarrays to manipulation of single reporter molecules. TIRFM: Total‐internal‐reflection fluorescence microscopy.

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Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study

et al. 2011

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The two-component regulatory system PhoR/PhoB induces the expression of several genes in response to phosphate starvation in Escherichia coli. In order to quantify these protein-DNA interactions and to study the time-resolved dynamics of the binding mechanism, the specific recognition of different oligonucleotide duplexes by the DNA-binding domain of PhoB (PhoB(DBD)) was analyzed using surface plasmon resonance. In addition the two point mutants PhoB(DBD)D196A and PhoB(DBD)R219A were obtained and the DNA recognition in comparison to the wildtype PhoB(DBD) was investigated. Aspartic acid 196 and arginine 219 mediate specific minor groove interactions. All results reveal that at high PhoB(DBD)-concentrations all recognition sequences of the pho box are occupied. Decreasing the protein amount results in a mixture of free oligonucleotides and DNA molecules occupied by two WT-PhoB(DBD). Moreover, the SPR results indicate that both binding site segments, the TGTCA-motif and the A/T-rich minor groove, are essential for the binding process. A comparison of different regulons additionally proved the dependency of the recognition process on the base composition of the minor groove.

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

Cited by 3 publications

References 34 publications

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

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

Fluorescence‐Emission Control of Single CdSe Nanocrystals Using Gold‐Modified AFM Tips

Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study

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