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

Abstract

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.

A single‐molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials

“…The planned configuration of SMFS assays consisted of DXS‐functionalized AFM cantilevers and pyruvate‐or G3P‐functionalized surfaces via heterobifunctional linkers (32). Such strategy introduces a distance between interacting molecules and surfaces, adds steric flexibility for the binding partners, and guarantees an almost complete reduction of unspecific binding events (53), although it might affect the calculation of the apparent kinetic and thermodynamic enzymatic parameters (23). Because E. coli DXS has 36 lysines, of which only K289 is present in the active center (54), we followed established protocols (48) for the covalent linkage of the enzyme to SiN 3 cantilevers through the lateral amino group in lysine residues, using a monodisperse ∼100 nm‐long polyethylene glycol (PEG) linker ( Fig.…”

Hidden Multiple Bond Effects in Dynamic Force Spectroscopy

“…(1)(2)(3). Typical examples include ligand-receptor compounds like antibodyantigen (6), protein-DNA (7), or supramolecular complexes (8). In all these experiments, one of the participating molecules is connected to a tiny force probe and the other to a substrate surface (or an immobilized micron-sized bead).…”

“…The depicted experimental data in (A)-(D) for a protein-DNA complex (PhoB mutant and target sequence, pulling velocity of 2000 nm/s, cantilever stiffness 13 pN/nm, linker length 30 nm) have been kindly provided by A. Bieker and D. Anselmetti (Bielefeld University). (E) and (F) Typical histograms of experimental rupture force distributions for two different pulling velocities v(7). After eliminating all the experimental force extension curves with clearly visible multiple bond signatures as those in (B) and (C), the number of remaining "apparent single bond rupture events" contributing to (E) was 202, and 151 for (F).…”

Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance

“…Moreover, unspecific background binding leading to false positives is another issue in these assays [2, 3]. In label-free approaches like atomic force microscopy-dynamic force spectroscopy experiments [4, 5], acoustic biosensors based on quartz crystal resonators [6], calorimetric biosensors [7], and surface plasmon resonance (SPR) inherently properties (e.g., mass) of the interacting molecules are measured. Therefore, these techniques avoid labeling steps and the disadvantages mentioned above.…”