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The aquation reactions of four rhenium(I) dicarbonyl complexes, [Re(CO)2(NN)(PR3)(Cl)], where NN = 1,10-phenanthroline (Phen) and 2,9-dimethyl-1,10-phenanthroline (DMPhen) and PR3 = 1,3,5-triaza-7-phosphaadamantane (PTA) and 1,4-diacetyl-1,3,7-triaza-5-phosphabicylco[3.3.1]nonane (DAPTA). Additionally, the anation reactions of the corresponding aqua complexes with Cl- were investigated. Single crystals of [Re(CO)2(DMPhen)(PTA)(Cl)]·DMF and [Re(CO)2(DMPhen)(DAPTA)(Cl)] were obtained, and their structures were determined using X-ray diffraction. The Re-Cl interatomic distances are 2.4991(13) and 2.4922(6) A, respectively, indicating a mild trans influence effect of the phosphine ligands. The rate constants, kaq, for the aquation reactions of these complexes spanned a range of (3.7 ± 0.3) × 10-4 to (15.7 ± 0.3) × 10-4 s-1 with the two Phen complexes having rate constants that are 2.5 times greater than those of the DMPhen complexes at 298 K. Similarly, the second-order anation rate constants (kCl) of the resulting aqua complexes, [Re(CO)2(NN)(PR3)(H2O)]+, with Cl- ions at 298 K varied between (2.99 ± 0.05) × 10-3 and (6.79 ± 0.09) × 10-3 M-1 s-1. Likewise, these rate constants for the Phen complexes were almost 2 times faster than those of the DMPhen complexes. The pKa values of the four aqua complexes were determined to be greater than 9.0 for all of the complexes with [Re(CO)2(Phen)(PTA)(H2O)]+ having the highest pKa value of 9.28 ± 0.03. From the pKa values and the ratios of the aquation and anation rate contants, which give thermodynamic Cl- binding constants, the speciation of the rhenium(I) complexes in blood plasma, the cytoplasm, and the cell nucleus were estimated. The data suggest that the aqua complexes would be the dominant species in all three environments. This result may have important implications on the potential biological activity of these complexes.

A range of N,O-donor atom salicylidene complexes of the type fac-[M(O,N-Bid)(CO)3(L)](n) (O,N-Bid = anionic N,O-bidentate ligands; L = neutral coordinated ligand) have been studied. The unique feature of the complexes which crystallize in a monoclinic isostructural space group for complexes containing methanol in the sixth position (L = MeOH) is highlighted. The reactivity and stability of the complexes were evaluated by rapid stopped-flow techniques, and the methanol substitution by a range of pyridine type ligands indicates significant activation by the N,O-salicylidene type of bidentate ligands as observed from the variation in the second-order rate constants. In particular, following the introduction of the sterically demanding and electron rich cyclohexyl salicylidene moiety on the bidentate ligand, novel limiting kinetic behavior is displayed by all entering ligands, thus enabling a systematic probe and manipulation of the limiting kinetic constants. Clear evidence of an interchange type of intimate mechanism for the methanol substitution is produced. The equilibrium and rate constants (25 °C) for the two steps in the dissociative interchange mechanism for methanol substitution in fac-[Re(Sal-Cy)(CO)3(MeOH)] (5) by the pyridine type ligands 3-chloropyridine, pyridine, 4-picoline, and DMAP are k3 (s(-1)), 40 ± 4, 13 ± 2, 10.4 ± 0.7, and 2.11 ± 0.09, and K2 (M(-1)), 0.13 ± 0.01, 0.21 ± 0.03, 0.26 ± 0.02, and 1.8 ± 0.1, respectively.

A range of fac-[Re(CO)3(L,L′-Bid)(H2O)]n (L,L′-Bid = neutral or monoanionic bidentate ligands with varied L,L′ donor atoms, N,N′, N,O, or O,O′: 1,10-phenanthroline, 2,2′-bipydine, 2-picolinate, 2-quinolinate, 2,4-dipicolinate, 2,4-diquinolinate, tribromotropolonate, and hydroxyflavonate; n = 0, +1) has been synthesized and the aqua/methanol substitution has been investigated. The complexes were characterized by UV–vis, IR and NMR spectroscopy and X-ray crystallographic studies of the compounds fac-[Re(CO)3(Phen)(H2O)]NO3·0.5Phen, fac-[Re(CO)3(2,4-dQuinH)(H2O)]·H2O, fac-[Re(CO)3(2,4-dQuinH)Py]Py, and fac-[Re(CO)3(Flav)(CH3OH)]·CH3OH are reported. A four order-of-magnitude of activation for the methanol substitution is induced as manifested by the second order rate constants with (N,N′-Bid) < (N,O-Bid) < (O,O′-Bid). Forward and reverse rate and stability constants from slow and stopped-flow UV/vis measurements (k1, M–1 s–1; k–1, s–1; K1, M–1) for bromide anions as entering nucleophile are as follows: fac-[R...

Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Förster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.

To stabilize air-water interfaces, as in foams, the adsorption of surface-active components is a prerequisite. An approach to controlling the surface activity of proteins is noncovalent complex formation with a polyelectrolyte in the bulk phase. The molecular properties of egg white ovalbumin in a complex with pectin in the bulk solution and at air/water interfaces were studied using drop tensiometry (ADT) and time-resolved fluorescence anisotropy techniques. The complex formation of ovalbumin with pectin in the bulk resulted in the formation of a compact structure with a different spatial arrangement depending on the protein/pectin ratio. Complex formation did not provide an altered protein structure, whereas the conformational stability was slightly increased in the complex. In excess pectin, an overall condensed complex structure is formed, whereas at limited pectin concentrations the structure of the complex is more "segmental" . The characteristics of these structures did not depend on pH in the 7.0 to 4.5 regime. Interaction with pectin in the bulk solution resulted in a significantly slower adsorption of the protein to the air/water interface. The limited mobility of the protein at the interface was found for both ovalbumin and ovalbumin-pectin complexes. From both the rotational dynamics and total fluorescence properties of the protein in the absence and presence of pectin, it was suggested that the complex does not dissociate at the interface. Ovalbumin in the complex retains its initial "aqueous" microenvironment at the interface, whereas in the absence of pectin the microenvironment of the protein changed to a more nonpolar one. This work illustrates a more general property of polyelectrolytes, namely, the ability to retain a protein in its microenvironment. Insight into this property provides a new tool for better control of the surface activity of complex biopolymer systems. © 2007 American Chemical Society.

Little is known about the changes in protein conformation that occur after displacement of a protein from an interface. Here, results are presented that give insight into the conformation of bovine a-lactalbumin (BLA) molecules that are displaced from a hydrophobic polystyrene interface. After the BLA molecules are adsorbed on polystyrene nanospheres, they are displaced from these nanospheres using two surfactants: Tween 20 and CHAPS. The properties of displaced BLA depend on the concentration of the surfactant used to displace the protein and on the incubation time during displacement, as can be concluded from intrinsic fluorescence spectroscopy, circular dichroism spectroscopy, and nondenaturing gel electrophoresis. CHAPS is more effective in displacing adsorbed BLA than Tween 20. The largest amount of displaced BLA (90% recovery) is obtained at a CHAPS concentration of 2 mM or higher. At a surfactant concentration of 1 or 2 mM, displaced BLA contains calcium and has native spectroscopic properties, indicating that BLA, which has a molten globulelike conformation in the adsorbed state, refolds to its native state upon displacement from the surface. However, non-native properties of displaced BLA are observed at a low surfactant concentration (0.3 mM) after prolonged incubation times. Under these conditions, the ensemble of displaced BLA molecules contains calcium, has a nativelike secondary structure, has a non-native tertiary structure, and contains a population of molecules that has a higher electrophoretic mobility on nondenaturing gels compared to that of native BLA. Intramolecular disulfide shuffling can cause the observed conformational changes. The disulfide shuffling is initiated by a few reactive groups on the surface of the nanospheres. It occurs during the homomolecular exchange of proteins at a surfactant concentration of 0.3 mM and is time dependent. Both Tween 20 and CHAPS are good candidates for the removal of proteins from interfaces, as long as the incubation time is short and the surfactant concentration is above a certain threshold. The displacement procedure presented here is essential for the future study of the atomic details of the conformation of proteins adsorbed on interfaces using NMR spectroscopy in combination with H/D exchange measurements.

A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pHor =6 and "folded" at pH5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.

The diffusion coefficients of 1-palmitoyl-2-(pyrenodecanoyl)-sn-glycero-3-phosphocholine (pyr10PC) in different bilayer membranes are determined from global analysis of the time-resolved fluorescence of the pyrene monomer and excimer emissions. The global kinetic model involves a time-dependent reaction rate coefficient derived from the analytical solution of the diffusion equation for bimolecular reactions in the two-dimensional surface of the membrane. The bilayer membranes are single unilamellar vesicles (SUV's or liposomes) composed of dioleoylphosphatidylcholine (DOPC), 1-stearoyl-2-arachidonoylphosphatidylcholine (SAPC), or dipalmitoylphosphatidylcholine (DPPC). All SUV's except the one composed of DPPC are in the liquid crystalline state. Liposomes composed of mixtures of DOPC and SAPC in various molar ratios are also investigated by the pyrene excimer reaction. The diffusion constant becomes larger when the fraction of SAPC in the mixed bilayers increases, indicating an apparent increase in fluidity. The latter observation is supported by photostationary measurements of the pyrene excimer to monomer intensity ratios of the same liposomes.

The dynamic properties of two BODIPY labeled phospholipid molecules in micellar systems have been studied with two different spectroscopic techniques, which provide complementary information (fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA), respectively). One phospholipid had the fluorescent probe attached to the headgroup (abbreviated as head-labeled) and the other one had the probe at the acyl chain at the sn-2 position of the phospholipid (abbreviated as tail-labeled). From FCS experiments, the translational diffusion constant, aggregation number and hydrodynamic size of the micelles were determined. The micelles were larger in size than reported in the literature, which is due to the incorporation of the relatively large sized fluorescent probe. The FCS results were confirmed by the results of dynamic light scattering experiments on both empty micelles and micelles containing the fluorescent lipid probe. Both results showed that the probe has a very signifi...

Quantitation of muscarinic receptors in the lungs in vivo with positron emission tomography (PET) is of clinical interest. For that purpose we decided to develop [C-11]-labeled ligands with a high affinity (K-D

The biologically active form of interferon gamma is a dimer composed of two noncovalently bound identical polypeptide chains of 17 kDa each. In this study, it was found that dissociation of the dimer into monomers significantly reduced the fluorescence quantum yield and the efficiency of the intermolecular Tyr to Trp radiationless energy transfer. The same process caused significant changes in the fluorescence decay and in the fluorescence anisotropy decay. The kinetic and thermodynamic parameters of the dimer-monomer equilibrium were determined by fluorescence measurements at different temperatures and by a theoretical mathematical model. Dissociation of the dimers into monomers was an endothermic process and was favored by concentrations of the protein lower than 1 microM and by increasing the temperature. It was accompanied by formation of aggregates, a slow and partially reversible process leading to inactivation of the interferon. It is suggested that certain monomeric conformers are competent for aggregation.

Transbilayer movement of the fluorescent membrane probe TMA-DPH [1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene] in the plasma membrane of human platelets was investigated by measuring fluorescence intensity and fluorescence decay. Labeling of unstimulated platelets by TMA-DPH results in a rapid increase in fluorescence intensity, leveling off within 1 min. Dilution of platelets into buffer without TMA-DPH leads to an almost complete rapid efflux of TMA-DPH, indicating that TMA-DPH labels only the outer leaflet of the plasma membrane. Transbilayer movement of the fluorescent probe in unstimulated platelets could be observed upon prolonged incubation and occurs with a t1/2 of 60-90 min. Stimulation of platelets with thrombin directly after the initial rapid uptake of TMA-DPH results in a fast increase in membrane-bound TMA-DPH, fully explained by the increase in plasma membrane caused by secretion of intracellular storage organelles. No indications for increased transbilayer movement of the probe were found, since dilution of thrombin-stimulated TMA-DPH-labeled platelets into buffer without TMA-DPH indicated no uptake of TMA-DPH by intracellular membranes. In contrast to thrombin, stimulation of TMA-DPH-labeled platelets with the Ca2(+)-ionophore ionomycin results in a much larger increase in fluorescence intensity. This process is accompanied by labeling of intracellular membranes as indicated by incomplete efflux of TMA-DPH after dilution of the stimulated platelets. Thus, stimulation of platelets by ionomycin gives rise to rapid and massive inward movement of TMA-DPH (t1/2 approximately 10-12 s). Prolonged incubation of platelets in the absence of any stimulus allows labeling of the total lipid pool, including intracellular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Microwave treatment combined with manipulation of the ionic strength of the reaction mixture increases the radiochemical yield dramatically of N-[F-18]fluoroalkylations of secondary amines as compared to thermal heating.

The spectral properties are compared for two 6,7-dimethyl-8-ribityllumazine proteins from marine bioluminescent bacteria, one from a psychrophile, Photobacterium phosphoreum, and the other from a thermophile, Photobacterium leiognathi. The visible spectral properties, which are the ones by which the protein performs its biological function of bioluminescence emission, are almost the same for the two proteins: at 2 degrees C and 50 mM Pi, pH 7, fluorescence quantum yield phi F = 0.59 and 0.54, respectively; fluorescence lifetime tau = 14.4 and 14.8 ns, respectively; fluorescence maxima, both 475 nm; absorption maximum, 417 and 420 nm, respectively; circular dichroism minima at around 420 nm, both -41 X 10(3) deg cm2 dmol-1. The ligand binding sites therefore must provide very similar environments, and arguments are presented that the bound ligand is relatively exposed to solvent. The dissociation equilibrium was studied by steady-state fluorescence polarization. The thermophilic protein binds the ligand with Kd (20 degrees C) = 0.016 microM, 10 times more tightly than the other protein [Kd (20 degrees C) = 0.16 microM]. The origin of the binding difference probably resides in differences in secondary structure. The tryptophan fluorescence spectra of the two proteins are different, but more significant is an observation of the decay of the tryptophan emission anisotropy. For the psychrophilic lumazine protein this anisotropy decays to zero in 1 ns, implying that its single tryptophan residue lies in a very "floppy" region of the protein. For the other protein, the anisotropy exhibits both a fast component and a slow one corresponding to rotation of the protein as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)

Photosensitized reduction of methyl viologen (MV/sup 2 +/) by 2 x 10/sup -4/ 10-methyl-5-deazaisoalloxazine-3-propanesulfonic acid (dF) in the presence of 1 x 10/sup -3/-10 x 10/sup -3/ M EDTA has been investigated in aqueous solutions by nanosecond laser flash photolysis. At substoichiometric concentrations of MV/sup 2 +/, the reactions /sup 1/dF (h nu)..-->.. /sup 1/dF* (isc)..-->.. /sup 3/dF*; /sup 3/dF* + EDTA ..-->.. /sup 2/dF. + EDTA/sup +/; /sup 2/dF. + MV/sup 2 +/ ..-->.. /sup 1/dF + MV/sup +/. have been discerned; thus dF acts as an electron relay. At higher MV/sup 2 +/ concentrations additional modes of dF catalyzed MV/sup +/. formation become available. 5 figures, 2 tables.

Horse heart cytochrome c and cytochrome c/sub 3/, isolated from Desulfovibrio vulgaris, have been incorporated in sodium bis(2-ethylhexyl)sulfosuccinate (AOT) entrapped water pools in heptane. The absorption spectra of the cytochromes have been found to be strongly dependent on the water to AOT concentration ratios. The proteins solubilized in heptane by the AOT reversed micelles have retained their ability to mediate electron transfer. They reacted very rapidly with hydrated electrons, generated pulse radiolytically or, alternatively, formed in the laser photoionization of pyrene.

In the synthesis of methanol on a CuO-ZnO-SiO/sub 2/ catalyst both the zinc oxide content in the catalyst and the presence of hydrogen influence the adsorption of CO on copper under reaction conditions; hydrogen adsorption on zinc probably inhibits the adsorption of CO. In a series of CuO-ZnO-SiO/sub 2/ catalysts with varying CuO/ZnO ratio, the maximum intensity of the infrared CO absorption band in a CO/H/sub 2/ atmosphere and the optimum activity for methanol synthesis coincide.