Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions.

Agrobacterium tumefaciens translocates T-DNA through a polar VirB/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T-DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T-DNA transfer by stimulating generation of multiple copies per cell of the T-DNA substrate (T-complex) through pairwise interactions with the processing factors VirD2 relaxase, VirC2, and VirD1. VirC1 also associates with the polar membrane and recruits T-complexes to cell poles, the site of VirB/D4 T4S machine assembly. VirC1 Walker A mutations abrogate T-complex generation and polar recruitment, whereas the native protein recruits T-complexes to cell poles independently of other polar processing factors (VirC2, VirD1) or T4S components (VirD4 substrate receptor, VirB channel subunits). We propose that A. tumefaciens has appropriated a progenitor ParA/MinD-like ATPase to promote conjugative DNA transfer by: (i) nucleating relaxosome assembly at oriT-like T-DNA border sequences and (ii) spatially positioning the transfer intermediate at the cell pole to coordinate substrate-T4S channel docking.

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.

Femtosecond dynamics of the forbidden carotenoid S1 state in light-harvesting complexes of purple bacteria observed after two-photon excitation

Time-resolved excited-state absorption intensities after direct two-photon excitation of the carotenoid S1 state are reported for light-harvesting complexes of purple bacteria. Direct excitation of the carotenoid S1 state enables the measurement of subsequent dynamics on a fs time scale without interference from higher excited states, such as the optically allowed S2 state or the recently discovered dark state situated between S1 and S2. The lifetimes of the carotenoid S1 states in the B800-B850 complex and B800-B820 complex of Rhodopseudomonas acidophila are 7 ± 0.5 ps and 6 ± 0.5 ps, respectively, and in the light-harvesting complex 2 of Rhodobacter sphaeroides ≈1.9 ± 0.5 ps. These results explain the differences in the carotenoid to bacteriochlorophyll energy transfer efficiency after S2 excitation. In Rps. acidophila the carotenoid S1 to bacteriochlorophyll energy transfer is found to be quite inefficient (φET1 <28%) whereas in Rb. sphaeroides this energy transfer is very efficient (φET1 ≈80%). The results are rationalized by calculations of the ensemble averaged time constants. We find that the Car S1 → B800 electronic energy transfer (EET) pathway (≈85%) dominates over Car S1 → B850 EET (≈15%) in Rb. sphaeroides, whereas in Rps. acidophila the Car S1 → B850 EET (≈60%) is more efficient than the Car S1 → B800 EET (≈40%). The individual electronic couplings for the Car S1 → BChl energy transfer are estimated to be approximately 5–26 cm−1. A major contribution to the difference between the energy transfer efficiencies can be explained by different Car S1 energy gaps in the two species.

Dynamical principles in biological processes: A model of charge migration in proteins and DNA

The generalized master equations (GMEs) that contain multiple time scales have been derived quantum mechanically. The GME method has then been applied to a model of charge migration in proteins that invokes the hole hopping between local amino acid sites driven by the torsional motions of the floppy backbones. This model is then applied to analyze the experimental results for sequence-dependent long-range hole transport in DNA reported by Meggers et al. [Meggers, E., Michel-Beyerle, M. E., & Giese, B. (1998) J. Am. Chem. Soc. 120, 12950–12955]. The model has also been applied to analyze the experimental results of femtosecond dynamics of DNA-mediated electron transfer reported by Zewail and co-workers [Wan, C., Fiebig, T., Kelley, S. O., Treadway, C. R., Barton, J. K. & Zewail, A. H. (1999) Proc. Natl. Acad. Sci. USA 96, 6014–6019]. The initial events in the dynamics of protein folding have begun to attract attention. The GME obtained in this paper will be applicable to this problem.

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 1010-fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (Onu) directly attacks PPi with a pKa vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and Onu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.

Dynamics of Fos-Jun-NFAT1 complexes

Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes.

On the role of the K-proton transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the “peroxy” state, Pr) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH ≈ 7.5. The movement of the Lys is proposed to regulate proton transfer by “shutting off” the protonic connectivity through the K-pathway after initiation of the O2 reduction chemistry. This “shutoff” prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST–Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST–Z2 is able to condense 130–150 kb bacterial artificial chromosomes (BACs) into protein–DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein–BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR– cells by GST–Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST–Z2-mediated gene transfer. Because DNA condensation by GST–Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.

Evidence for the role of proteoglycans in cation-mediated gene transfer.

We report evidence that gene complexes, consisting of polycations and plasmid DNA enter cells via binding to membrane-associated proteoglycans. Treatment of HeLa cells with sodium chlorate, a potent inhibitor of proteoglycan sulfation, reduced luciferase expression by 69%. Cellular treatment with heparinase and chondroitinase ABC inhibited expression by 78% and 20% with respect to control cells. Transfection was dramatically inhibited by heparin and heparan sulfate and to a smaller extent by chondroitan sulfate B. Transfection of mutant, proteoglycan deficient Chinese hamster ovary cells was 53 x lower than of wild-type cells. For each of these assays, the intracellular uptake of DNA at 37 degrees C and the binding of DNA to the cell membrane at 4 degrees C was impaired. Preliminary transfection experiments conducted in mutant and wild-type Chinese hamster ovary cells suggest that transfection by some cationic lipids is also proteoglycan dependent. The variable distribution of proteoglycans among tissues may explain why some cell types are more susceptible to transfection than others.

Mechanism of oligonucleotide release from cationic liposomes.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.

Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers.

Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines.