Due to a high density of negative charges on its surface, DNA condenses cations as counterions, forming the so-called “ion atmosphere”. Although the release of counterions upon DNA–protein association has been postulated to have a major contribution to the binding thermodynamics, this release remains to be confirmed through a direct observation of the ions. Herein, we report the characterization of the ion atmosphere around DNA using NMR spectroscopy and directly detect the release of counterions upon DNA–protein association. NMR-based diffusion data reveal the highly dynamic nature of counterions within the ion atmosphere around DNA. Counterion release is observed as an increase in the apparent ionic diffusion coefficient, which directly provides the number of counterions released upon DNA–protein association. 相似文献
The diffusion coefficient of the self-assembled supramolecular cluster [Ga4L6]12- depends on the cationic counterions in solution. Diffusion coefficients were determined using the pulsed-gradient spin-echo 1H NMR method and fit using nonlinear least-squares refinement. Saturation studies revealed a small number of ion-association sites on the exterior of the assembly and the direct observation of ion association in water. The addition of excess alkali-metal cations displaces the ion-associated hydrophobic tetra-alkyl-ammonium cations. Comparisons between tetraethyl- and tetra-propyl-ammonium show a preference for ion association with the more hydrophobic cation. 相似文献
We report herein an interesting dynamic translocation process of countercations around one polyoxometalate(POM)–organic hybrid anionic cluster at various concentrations and temperatures. It was found that both electrostatic interactions and cation–π interactions regulate the position of small countercations around single clusters. The dynamic geometry and the symmetry of the hybrid macroions are largely affected by the type of counterions, as shown by nuclear magnetic resonance (NMR) spectroscopy studies and all‐atom molecular dynamics simulation. It is also shown that electrostatic interactions dominate over cation–π interactions in determining the locations of the counterions in the current system. 相似文献
Hoogsteen DNA base pairs (bps) are an alternative base pairing to canonical Watson–Crick bps and are thought to play important biochemical roles. Hoogsteen bps have been reported in a handful of X‐ray structures of protein–DNA complexes. However, there are several examples of Hoogsteen bps in crystal structures that form Watson–Crick bps when examined under solution conditions. Furthermore, Hoogsteen bps can sometimes be difficult to resolve in DNA:protein complexes by X‐ray crystallography due to ambiguous electron density and by solution‐state NMR spectroscopy due to size limitations. Here, using infrared spectroscopy, we report the first direct solution‐state observation of a Hoogsteen (G–C+) bp in a DNA:protein complex under solution conditions with specific application to DNA‐bound TATA‐box binding protein. These results support a previous assignment of a G–C+ Hoogsteen bp in the complex, and indicate that Hoogsteen bps do indeed exist under solution conditions in DNA:protein complexes. 相似文献
In this work, a novel NMR method for the identification of preferential coordination sites between physiologically relevant counterions and nucleic acid bases is demonstrated. In this approach, the NMR cross-correlated relaxation rates between the aromatic carbon chemical shift anisotropy and the proton-carbon dipolar interaction are monitored as a function of increasing Na(+), K(+), and Mg(2+) concentrations. Increasing the counterion concentration modulates the residence times of the counterions at specific sites around the nucleic acid bases. It is demonstrated that the modulation of the counterion concentration leads to sizable variations of the cross-correlated relaxation rates, which can be used to probe the site-specific counterion coordination. In parallel, the very same measurements report on the rotational tumbling of DNA, which, as shown here, depends on the nature of the ion and its concentration. This methodology is highly sensitive and easily implemented. The method can be used to cross-validate and/or complement direct but artifact-prone experimental techniques such as X-ray diffraction, NMR analysis with substitutionary ions, and molecular dynamics simulations. The feasibility of this technique is demonstrated on the extraordinarily stable DNA mini-hairpin d(GCGAAGC). 相似文献
Distamycin‐based tetrapeptide ( 1 ) was covalently tethered to both ends of the central dihydroxyazobenzene moiety at either the 2,2′ or 4,4′ positions. This afforded two isomeric, distamycin–azobenzene–distamycin systems, 2 (para) and 3 (ortho), both of them being photoisomerizable. Illumination of these conjugates in solution at approximately 360 nm induced photoisomerization and the time course of the process was followed by UV/Vis and 1H NMR spectroscopy. The kinetics of the thermal reversion at various temperatures of cis to trans isomers of the conjugates obtained after photoillumination were also examined. This afforded the respective thermal‐activation parameters. Both the molecular architecture and the location of the substituent around the core azobenzene determined the rate and activation‐energy barrier for the cis‐to‐trans back‐isomerization of these conjugates in solution. Duplex–DNA binding of the conjugates and the changes in DNA‐binding efficiency upon photoisomerization was also examined by CD spectroscopy, thermal denaturation studies, and a Hoechst displacement assay. The conjugate 2 showed higher DNA‐binding affinity and a greater change in the DNA‐binding efficiency upon photoisomerization compared with its 2,2′‐disubstituted counterpart. The experimental findings were substantiated by using molecular‐docking studies involving each conjugate with a model duplex d[(GC(AT)10CG)]2 DNA molecule. 相似文献
In recent years, significant progress has been made towards uncovering the physical mechanisms of low‐hydration polymorphism in double‐helical DNA. The effect appears to be mechanistically similar in different biological systems, and it is due to the ability of water to form spanning H‐bonded networks around biomacromolecules via a quasi‐two‐dimensional percolation transition. In the case of DNA, disintegration of the spanning H‐bonded network leads to electrostatic condensation of DNA strands because, below the percolation threshold, water loses its high dielectric permittivity, whereas the concentration of neutralizing counterions becomes high. In this Concept article arguments propose that this simple electrostatic mechanism represents the universal origin of low‐hydration polymorphism in DNA. 相似文献
The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering. 相似文献
The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering. 相似文献
DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. 13C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues. 相似文献
Rod-like polyelectrolytes are an interesting model system because their persistence length is independent of the ionic strength and pH of the surrounding medium and they permit the investigation of polyelectrolytes in the absence of conformational degrees of freedom. In this work, rigid-rod poly(aramide) polyelectrolytes were synthesized by the Higashi method. Electrophoresis NMR spectroscopy in conjunction with diffusion NMR spectroscopy has been applied to determine the effective charge of the polymer. The charge was determined from the balance between the force in the electric field and the hydrodynamic friction in the steady-state electrophoretic motion. Because only organic counterions were present, and were identified in the proton NMR spectra, the counterions were investigated as well, and the fraction of condensed counterions was determined directly. From the effective charge per molecule and the knowledge of the fraction of condensed counterions, the total charge per molecule was determined. Finally, from the total charge, the number of repeat units and thus the molecular weight were inferred. 相似文献
A new cationic poly(3,4‐ethylenedioxythiophene)–DNA composite (P(EDOT‐N)‐DNA) has been prepared by in situ chemical oxidative polymerization of EDOT‐N monomer in the presence of salmon DNA as template. Scanning electron microscopy shows that the P(EDOT‐N)–DNA composite forms a porous pattern with a high surface area that is favorable for ion diffusion throughout the materials, which leads to improved capacitive activity. The P(EDOT‐N)–DNA composite exhibits superior capacitance behavior as well as good charge–discharge reversibility. The P(EDOT‐N)–DNA composite shows low cytotoxicity to living cells even at a concentration of 300 mg · L−1. The good biocompatibility of P(EDOT‐N)–DNA imparts good potential as an environmentally friendly electrode material for energy storage devices, even in a biological environment owing to the combination of DNA with conjugated polymers.
Despite their roles in controlling many cellular processes, weak and transient interactions between large structured macromolecules and disordered protein segments cannot currently be characterized at atomic resolution by X‐ray crystallography or solution NMR. Solid‐state NMR does not suffer from the molecular size limitations affecting solution NMR, and it can be applied to molecules in different aggregation states, including non‐crystalline precipitates and sediments. A solid‐state NMR approach based on high magnetic fields, fast magic‐angle sample spinning, and deuteration provides chemical‐shift and relaxation mapping that enabled the characterization of the structure and dynamics of the transient association between two regions in an 80 kDa protein assembly. This led to direct verification of a mechanism of regulation of E. coli DNA metabolism. 相似文献
In this study, DNA local structures with bulged bases and mismatched base pairs as well as ordinary full‐matched base pairs by using 19F NMR spectroscopy with 19F‐labeled oligodeoxynucleotides (ODNs) were monitored. The chemical shift change in the 19F NMR spectra allowed discrimination of the DNA structures. Two types of ODNs possessing the bis(trifluoromethyl)benzene unit (F‐unit) at specified uridines were prepared and hybridized with their complementary or noncomplementary strands to form matched, mismatched, or bulged duplexes. By using ODN F1, in which an F‐unit was connected directly to a propargyl amine‐substituted uridine, three local structures, that is, full‐matched, G–U mismatch, and A‐bulge could be analyzed, whereas other structures could not be discriminated. A molecular modeling study revealed that the F‐unit in ODN F1 interacted little with the nucleobases and sugar backbone of the opposite strand because the linker length between the F‐unit and the uridine base was too short. Therefore, the capacity of ODN F1 to discriminate the DNA local structures was limited. Thus, ODN F2 was designed to improve this system; aminobenzoic acid was inserted between the F‐unit and uridine base so the F‐unit could interact more closely with the opposite strand. Eventually, the G‐bulge and T–U mismatch and the three aforementioned local structures could be discriminated by using ODN F2. In addition, the dissociation processes of these duplexes could be monitored concurrently by 19F NMR spectroscopy. 相似文献
In this paper we study the influence of the formation of intrachain ion pairs (salt bonds) and the distribution of counterions on the behavior of single polyampholyte chains in a dilute solution. It has been shown that neutral polyampholyte chains can undergo jump‐like collapse transition from the swollen state to the globular state with the formation of ion pairs between oppositely charged ions of the chain. A polyampholyte chain with an excess charge shows the behavior of a conventional polyelectrolyte chain and counterions play an important role in the chain behavior. We distinguish three possible states of counterions: free counterions inside and outside the macromolecule, and a bound state of counterions forming ion pairs with the corresponding ions of the polymer chain. We found a non‐monotonous behavior of the chain upon increasing the excess charge on the chain: the chain swells from a compact state to elongated conformation and shrinks again to the compact state when the excess charge of the chain is increased. 相似文献
Reverse Watson–Crick DNA with parallel‐strand orientation (ps DNA) has been constructed. Pyrrolo‐dC (PyrdC) nucleosides with phenyl and pyridinyl residues linked to the 6 position of the pyrrolo[2,3‐d]pyrimidine base have been incorporated in 12‐ and 25‐mer oligonucleotide duplexes and utilized as silver‐ion binding sites. Thermal‐stability studies on the parallel DNA strands demonstrated extremely strong silver‐ion binding and strongly enhanced duplex stability. Stoichiometric UV and fluorescence titration experiments verified that a single 2pyPyrdC–2pyPyrdC pair captures two silver ions in ps DNA. A structure for the PyrdC silver‐ion base pair that aligns 7‐deazapurine bases head‐to‐tail instead of head‐to‐head, as suggested for canonical DNA, is proposed. The silver DNA double helix represents the first example of a ps DNA structure built up of bidentate and tridentate reverse Watson–Crick base pairs stabilized by a dinuclear silver‐mediated PyrdC pair. 相似文献