Toward artificial developmental regulators

A polyamide-peptide conjugate is designed which recruits sequence specifically the developmental regulator Exd to a cognate DNA site. In particular, an eight-ring hairpin polyamide (Im-Im-Py(C3H6NHR)-Py-gamma-Im-Py-Py-Py-beta-Dp) with a heptapeptide (R = Ac-Phe-Tyr-Pro-Trp-Met-Lys-Gly-) attached on a central ring was shown to induce cooperative binding of the Drosophila Hox protein cofactor Exd with a Kd of 4.4 nM in vitro, an order of magnitude more efficient than the natural Hox protein partner Ubx. The conjugate joins two sequence specific domains, one for DNA and one for the protein. This small molecule thus serves as a cooperative protein-DNA dimerizer, which mimics the natural Hox family of developmental regulators.     

Scoring function based approach for locating binding sites and understanding recognition mechanism of protein-DNA complexes

Protein-DNA recognition plays an essential role in the regulation of gene expression. Understanding the recognition mechanism of protein-DNA complexes is a challenging task in molecular and computational biology. In this work, a scoring function based approach has been developed for identifying the binding sites and delineating the important residues for binding in protein-DNA complexes. This approach considers both the repulsive interactions and the effect of distance between atoms in protein and DNA. The results showed that positively charged, polar, and aromatic residues are important for binding. These residues influence the formation of electrostatic, hydrogen bonding, and stacking interactions. Our observation has been verified with experimental binding specificity of protein-DNA complexes and found to be in good agreement with experiments. The comparison of protein-RNA and protein-DNA complexes reveals that the contribution of phosphate atoms in DNA is twice as large as in protein-RNA complexes. Furthermore, we observed that the positively charged, polar, and aromatic residues serve as hotspot residues in protein-RNA complexes, whereas other residues also altered the binding specificity in protein-DNA complexes. Based on the results obtained in the present study and related reports, a plausible mechanism has been proposed for the recognition of protein-DNA complexes.     

Structural and Kinetic Profiling of Allosteric Modulation of Duplex DNA Induced by DNA-Binding Polyamide Analogues

A combined structural and quantitative biophysical profile of the DNA binding affinity, kinetics and sequence-selectivity of hairpin polyamide analogues is described. DNA duplexes containing either target polyamide binding sites or mismatch sequences are immobilized on a microelectrode surface. Quantitation of the DNA binding profile of polyamides containing N-terminal 1-alkylimidazole (Im) units exhibit picomolar binding affinities for their target sequences, whereas 5-alkylthiazole (Nt) units are an order of magnitude lower (low nanomolar). Comparative NMR structural analyses of the polyamide series shows that the steric bulk distal to the DNA-binding face of the hairpin iPr-Nt polyamide plays an influential role in the allosteric modulation of the overall DNA duplex structure. This combined kinetic and structural study provides a foundation to develop next-generation hairpin designs where the DNA-binding profile of polyamides is reconciled with their physicochemical properties.     

Characterization of nonspecific protein-DNA interactions by 1H paramagnetic relaxation enhancement

Nonspecific protein-DNA interactions play an important role in a variety of contexts related to DNA packaging, nucleoprotein complex formation, and gene regulation. Biophysical characterization of nonspecific protein-DNA interactions at the atomic level poses significant challenges owing to the dynamic nature of such complexes. Although NMR spectroscopy represents a powerful tool for the analysis of dynamic systems, conventional NMR techniques have provided little information on nonspecific protein-DNA interactions. We show that intermolecular (1)H paramagnetic relaxation enhancement (PRE) arising from Mn(2+) chelated to an EDTA-group covalently attached to a thymine base (dT-EDTA-Mn(2+)) in DNA provides a unique approach for probing the global dynamics and equilibrium distribution of nonspecific protein-DNA interactions. For nonspecific DNA binding, similar intermolecular (1)H-PRE profiles are observed on the (1)H resonances of the bound protein when dT-EDTA-Mn(2+) is located at either end of a DNA oligonucleotide duplex. We demonstrate the applicability of this approach to HMG-box proteins and contrast the results obtained for nonspecific DNA binding of the A-box of HMGB-1 (HMGB-1A) with sequence-specific DNA binding of the related SRY protein. Intermolecular (1)H-PRE data demonstrate unambiguously that HMGB-1A binds to multiple sites in multiple orientations even on a DNA fragment as short as 14 base pairs. Combining the (1)H-PRE data with the crystal structure of the HMGB-1 A-box/cisplatin-modified DNA complex allows one to obtain a semiquantitative estimate of the equilibrium populations at the various sites.     

Nonspecific protein-DNA interactions: complexation of alpha-chymotrypsin with a genomic DNA

In this contribution, we report studies on nonspecific protein-DNA interactions of an enzyme protein bovine pancreatic alpha-chymotrypsin (CHT) with genomic DNA (from salmon testes) using two biologically common fluorescent probes: 1-anilinonaphthalene-8-sulfonate (ANS) and 2,6-p-toluidinonaphthalene sulfonate (TNS). TNS molecules that are nonspecifically bound to positively charged basic residues at the surface sites, not in the hydrophobic cavities of the protein, are preferentially displaced upon complexation of TNS-labeled CHT with DNA. The time-resolved fluorescence anisotropy of TNS molecules bound to hydrophobic cavities/clefts of CHT reveals that global tumbling motion of the protein is almost frozen in the protein-DNA complex. A control study on TNS-labeled human serum albumin (HSA) upon interaction with DNA clearly indicates that the ligands in the deep pockets of the protein cannot be displaced by interaction with DNA. We have also found that ANS, which binds to a specific surface site of CHT, is not displaced by DNA. The intactness of the ANS binding in CHT upon complexation with DNA offers the opportunity to measure the distance between the ANS binding site and the contact point of the ethidium bromide (EB)-labeled DNA using the F?rster resonance energy transfer (FRET) technique. Enzymatic activity studies on CHT on a substrate (Ala-Ala-Phe 7-amido-4-methyl coumarin) reveal that the active site of the enzyme remains open for the substrate even in the protein-DNA complex. Circular dichroism (CD) studies on CHT upon complexation with DNA confirm the structural integrity of CHT in the complex. Our studies have attempted to explore an application of nonspecific protein-DNA interactions in the characterization of ligand binding of a protein in solution.     

Evaluation of binding selectivity of a polyamide probe to single base-pair different dna in A·T-rich region by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) was used for the evaluation of the binding selectivity of a polyamide probe to single-base pair different DNA in an A.T-rich region. In this procedure, DeltaIr(dsn) was introduced as a parameter to compare the binding affinities of the polyamides with the duplex DNA. The results show that ESI-MS is a very useful tool for analysis of binding selectivity of a polyamide probe to single-base pair different DNA.     

Synthesis of a Pair of Derivatives of N-Methylpyrrole Polyamide

A pair of derivatives of tetrameric N-methylpyrrole polyamide were synthesized in order to develop a new method for the study of interaction of the polyamide derivatives with DNA. Indole acetic acid and nicotinic acid were introduced to the polyamide in the synthesized compound, which showed an expected red shift in the UV spectrum. These compounds may function as a potential tool in the detection of the polyamide binding to DNA.     

A new H/D exchange- and mass spectrometry-based method for thermodynamic analysis of protein-DNA interactions

The application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the thermodynamic analysis of protein-DNA complexes is described. A series of five model protein-DNA complexes involving two known DNA binding proteins, Arc repressor and CopG, were analyzed in order to determine the accuracy, precision, and generality of the SUPREX technique for quantifying the strength of protein-DNA interactions. For protein-DNA complexes that reversibly unfold in a two-state manner, we demonstrate that reasonably precise Kd values in agreement with those determined by conventional techniques can be determined by SUPREX. In the case of protein-DNA complexes that are not well modeled by a two-state unfolding mechanism, we find that relative binding affinities can be determined in the SUPREX experiment.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.