Charge transfer and transport in DNA

We explore charge migration in DNA, advancing two distinct mechanisms of charge separation in a donor (d)–bridge ({Bj})–acceptor (a) system, where {Bj} = B1,B2, … , BN are the N-specific adjacent bases of B-DNA: (i) two-center unistep superexchange induced charge transfer, d*{Bj}a → d∓{Bj}a±, and (ii) multistep charge transport involves charge injection from d* (or d+) to {Bj}, charge hopping within {Bj}, and charge trapping by a. For off-resonance coupling, mechanism i prevails with the charge separation rate and yield exhibiting an exponential dependence ∝ exp(−βR) on the d-a distance (R). Resonance coupling results in mechanism ii with the charge separation lifetime τ ∝ Nη and yield Y ≃ (1 + δ̄ Nη)−1 exhibiting a weak (algebraic) N and distance dependence. The power parameter η is determined by charge hopping random walk. Energetic control of the charge migration mechanism is exerted by the energetics of the ion pair state d∓B1±B2 … BNa relative to the electronically excited donor doorway state d*B1B2 … BNa. The realization of charge separation via superexchange or hopping is determined by the base sequence within the bridge. Our energetic–dynamic relations, in conjunction with the energetic data for d*/d− and for B/B+, determine the realization of the two distinct mechanisms in different hole donor systems, establishing the conditions for “chemistry at a distance” after charge transport in DNA. The energetic control of the charge migration mechanisms attained by the sequence specificity of the bridge is universal for large molecular-scale systems, for proteins, and for DNA.

Long-time quantum simulation of the primary charge separation in bacterial photosynthesis.

Accurate quantum mechanical simulations of the primary charge transfer in photosynthetic reaction centers are reported. The process is modeled by three coupled electronic states corresponding to the photoexcited chlorophyll special pair (donor), the reduced bacteriopheophytin (acceptor), and the reduced accessory chlorophyll (bridge) that interact with a dissipative medium of protein and solvent degrees of freedom. The time evolution of the excited special pair is followed over 17 ps by using a fully quantum mechanical path integral scheme. We find that a free energy of the reduced accessory chlorophyll state approximately equal to 400 cm(-1) lower than that of the excited special pair state yields state populations in agreement with experimental results on wild-type and modified reaction centers. For this energetic configuration electron transfer is a two-step process.

The long-range supraorganization of the bacterial photosynthetic unit: A key role for PufX

Bacterial photosynthesis relies on the interplay between light harvesting and electron transfer complexes, all of which are located within the intracytoplasmic membrane. These complexes capture and transfer solar energy, which is used to generate a proton gradient. In this study, we identify one of the factors that determines the organization of these complexes. We undertook a comparison of the organization of the light-harvesting complex 1 (LH1)/reaction center (RC) cores in the LH2− mutant of Rhodobacter sphaeroides in the presence or absence of the PufX protein. From polarized absorption spectra on oriented membranes, we conclude that PufX induces a specific orientation of the reaction center in the LH1 ring, as well as the formation of a long-range regular array of LH1-RC cores in the photosynthetic membrane. From our data, we have constructed a precise model of how the RC is positioned within the LH1 ring relative to the long (orientation) axis of the photosynthetic membrane.

Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators.

Insulin promotes rapid delivery of N-methyl-d- aspartate receptors to the cell surface by exocytosis

Insulin potentiates N-methyl-d-aspartate receptors (NMDARs) in neurons and Xenopus oocytes expressing recombinant NMDARs. The present study shows that insulin induced (i) an increase in channel number times open probability (nPo) in outside-out patches excised from Xenopus oocytes, with no change in mean open time, unitary conductance, or reversal potential, indicating an increase in n and/or Po; (ii) an increase in charge transfer during block of NMDA-elicited currents by the open channel blocker MK-801, indicating increased number of functional NMDARs in the cell membrane with no change in Po; and (iii) increased NR1 surface expression, as indicated by Western blot analysis of surface proteins. Botulinum neurotoxin A greatly reduced insulin potentiation, indicating that insertion of new receptors occurs via SNARE-dependent exocytosis. Thus, insulin potentiation occurs via delivery of new channels to the plasma membrane. NMDARs assembled from mutant subunits lacking all known sites of tyrosine and serine/threonine phosphorylation in their carboxyl-terminal tails exhibited robust insulin potentiation, suggesting that insulin potentiation does not require direct phosphorylation of NMDAR subunits. Because insulin and insulin receptors are localized to glutamatergic synapses in the hippocampus, insulin-regulated trafficking of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

The CuA center of cytochrome-c oxidase: electronic structure and spectra of models compared to the properties of CuA domains.

The electronic structure and spectrum of several models of the binuclear metal site in soluble CuA domains of cytochrome-c oxidase have been calculated by the use of an extended version of the complete neglect of differential overlap/spectroscopic method. The experimental spectra have two strong transitions of nearly equal intensity around 500 nm and a near-IR transition close to 800 nm. The model that best reproduces these features consists of a dimer of two blue (type 1) copper centers, in which each Cu atom replaces the missing imidazole on the other Cu atom. Thus, both Cu atoms have one cysteine sulfur atom and one imidazole nitrogen atom as ligands, and there are no bridging ligands but a direct Cu-Cu bond. According to the calculations, the two strong bands in the visible region originate from exciton coupling of the dipoles of the two copper monomers, and the near-IR band is a charge-transfer transition between the two Cu atoms. The known amino acid sequence has been used to construct a molecular model of the CuA site by the use of a template and energy minimization. In this model, the two ligand cysteine residues are in one turn of an alpha-helix, whereas one ligand histidine is in a loop following this helix and the other one is in a beta-strand.

DNA packing in stable lipid complexes designed for gene transfer imitates DNA compaction in bacteriophage

The structure of complexes made from DNA and suitable lipids (lipoplex, Lx) was examined by cryo-electron microscopy (cryoEM). We observed a distinct concentric ring-like pattern with striated shells when using plasmid DNA. These spherical multilamellar particles have a mean diameter of 254 nm with repetitive spacing of 7.5 nm with striation of 5.3 nm width. Small angle x-ray scattering revealed repetitive ordering of 6.9 nm, suggesting a lamellar structure containing at least 12 layers. This concentric and lamellar structure with different packing regimes also was observed by cryoEM when using linear double-stranded DNA, single-stranded DNA, and oligodeoxynucleotides. DNA chains could be visualized in DNA/lipid complexes. Such specific supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of DNA in a liquid crystalline phase. CryoEM examination of T4 phage DNA packed either in T4 capsides or in lipidic particles showed similar patterns. Small angle x-ray scattering suggested an hexagonal phase in Lx-T4 DNA. Our results indicate that both lamellar and hexagonal phases may coexist in the same Lx preparation or particle and that transition between both phases may depend on equilibrium influenced by type and length of the DNA used.

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.

Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA–liposome complexes

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA–liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.

Femtosecond dynamics of DNA-mediated electron transfer

Diverse biophysical and biochemical studies have sought to understand electron transfer (ET) in DNA in part because of its importance to DNA damage and its repair. However, the dynamics and mechanisms of the elementary processes of ET in this medium are not fully understood and have been heavily debated. Two fundamental issues are the distance over which charge is transported and the time-scale on which the transport through the π-stack of the DNA base pairs may occur. With femtosecond resolution, we report direct observation in DNA of ultrafast ET, initiated by excitation of tethered ethidium (E), the intercalated electron acceptor (A); the electron donor (D) is 7-deazaguanine (Z), a modified base, placed at different, fixed distances from A. The ultrafast ET between these reactants in DNA has been observed with time constants of 5 ps and 75 ps and was found to be essentially independent of the D–A separation (10–17 Å). However, the ET efficiency does depend on the D–A distance. The 5-ps decay corresponds to direct ET observed from 7-deazaguanine but not guanine to E. From measurements of orientation anisotropies, we conclude that the slower 75-ps process requires the reorientation of E before ET, similar to E/nucleotide complexes in water. These results reveal the nature of ultrafast ET and its mechanism: in DNA, ET cannot be described as in proteins simply by a phenomenological parameter, β. Instead, the involvement of the base pairs controls the time scale and the degree of coherent transport.

Formation of stable cationic lipid/DNA complexes for gene transfer.

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

Fluorescence and photobleaching dynamics of single light-harvesting complexes

Single light-harvesting complexes LH-2 from Rhodopseudomonas acidophila were immobilized on various charged surfaces under physiological conditions. Polarized light experiments showed that the complexes were situated on the surface as nearly upright cylinders. Their fluorescence lifetimes and photobleaching properties were obtained by using a confocal fluorescence microscope with picosecond time resolution. Initially all molecules fluoresced with a lifetime of 1 ± 0.2 ns, similar to the bulk value. The photobleaching of one bacteriochlorophyll molecule from the 18-member assembly caused the fluorescence to switch off completely, because of trapping of the mobile excitations by energy transfer. This process was linear in light intensity. On continued irradiation the fluorescence often reappeared, but all molecules did not show the same behavior. Some LH-2 complexes displayed a variation of their quantum yields that was attributed to photoinduced confinement of the excited states and thereby a diminution of the superradiance. Others showed much shorter lifetimes caused by excitation energy traps that are only ≈3% efficient. On repeated excitation some molecules entered a noisy state where the fluorescence switched on and off with a correlation time of ≈0.1 s. About 490 molecules were examined.

Energy transfer to a proton-transfer fluorescence probe: Tryptophan to a flavonol in human serum albumin

A protein fluorescence probe system, coupling excited-state intermolecular Förster energy transfer and intramolecular proton transfer (PT), is presented. As an energy donor for this system, we used tryptophan, which transfers its excitation energy to 3-hydroxyflavone (3-HF) as a flavonol prototype, an acceptor exhibiting excited-state intramolecular PT. We demonstrate such a coupling in human serum albumin–3-HF complexes, excited via the single intrinsic tryptophan (Trp-214). Besides the PT tautomer fluorescence (λmax = 526 nm), these protein–probe complexes exhibit a 3-HF anion emission (λmax = 500 nm). Analysis of spectroscopic data leads to the conclusion that two binding sites are involved in the human serum albumin–3-HF interaction. The 3-HF molecule bound in the higher affinity binding site, located in the IIIA subdomain, has the association constant (k1) of 7.2 × 105 M−1 and predominantly exists as an anion. The lower affinity site (k2 = 2.5 × 105 M−1), situated in the IIA subdomain, is occupied by the neutral form of 3-HF (normal tautomer). Since Trp-214 is situated in the immediate vicinity of the 3-HF normal tautomer bound in the IIA subdomain, the intermolecular energy transfer for this donor/acceptor pair has a 100% efficiency and is followed by the PT tautomer fluorescence. Intermolecular energy transfer from the Trp-214 to the 3-HF anion bound in the IIIA subdomain is less efficient and has the rate of 1.61 × 108 s−1, thus giving for the donor/acceptor distance a value of 25.5 Å.

Determination of the binding sites of the proton transfer inhibitors Cd2+ and Zn2+ in bacterial reaction centers

The reaction center (RC) from Rhodobacter sphaeroides couples light-driven electron transfer to protonation of a bound quinone acceptor molecule, QB, within the RC. The binding of Cd2+ or Zn2+ has been previously shown to inhibit the rate of reduction and protonation of QB. We report here on the metal binding site, determined by x-ray diffraction at 2.5-Å resolution, obtained from RC crystals that were soaked in the presence of the metal. The structures were refined to R factors of 23% and 24% for the Cd2+ and Zn2+ complexes, respectively. Both metals bind to the same location, coordinating to Asp-H124, His-H126, and His-H128. The rate of electron transfer from QA− to QB was measured in the Cd2+-soaked crystal and found to be the same as in solution in the presence of Cd2+. In addition to the changes in the kinetics, a structural effect of Cd2+ on Glu-H173 was observed. This residue was well resolved in the x-ray structure—i.e., ordered—with Cd2+ bound to the RC, in contrast to its disordered state in the absence of Cd2+, which suggests that the mobility of Glu-H173 plays an important role in the rate of reduction of QB. The position of the Cd2+ and Zn2+ localizes the proton entry into the RC near Asp-H124, His-H126, and His-H128. Based on the location of the metal, likely pathways of proton transfer from the aqueous surface to QB⨪ are proposed.

Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn2+ or Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA−•QB−• + H+ → QA(QBH)−, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD ≤ 0.5 μM) and reduced the observed value of kAB(2) 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the kAB(2) reaction. In native RCs, kAB(2) was previously shown to be rate-limited by electron transfer based on the dependence of kAB(2) on the driving force for electron transfer. Upon addition of Zn2+ or Cd2+, kAB(2) became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 102-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+•QAQB−• → DQAQB suggests that the binding site is located far (>10 Å) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB−• occurs.