Fluorescence lifetime imaging microscopy and time-resolved anisotropy of nanomaterial-induced changes to red blood cell membranes.

With the use of engineered nano-materials (ENM) becoming more prevalent, it is essential to determine potential human health impacts. Specifically, the effects on biological lipid membranes will be important for determining molecular events that may contribute to both toxicity and suitable biomedical applications. To better understand the mechanisms of ENM-induced hemolysis and membrane permeability, fluorescence lifetime imaging microscopy (FLIM) was performed on human red blood cells (RBC) exposed to titanium dioxide ENM, zinc oxide ENM, or micron-sized crystalline silica. In the FLIM images, changes in the intensity-weighted fluorescence lifetime of the lipophilic fluorescence probe Di-4-ANEPPDHQ were used to identify localized changes to membrane. Time-resolved fluorescence anisotropy and FLIM of RBC treated with methyl-ß-cyclodextrin was performed to aid in interpreting how changes to membrane order influence changes in the fluorescence lifetime of the probe. Treatment of RBC with methyl-ß-cyclodextrin caused an increase in the wobble-in-a-cone angle and shorter fluorescence lifetimes of di-4-ANEPPDHQ. Treatment of RBC with titanium dioxide caused a significant increase in fluorescence lifetime compared to non-treated samples, indicating increased membrane order. Crystalline silica also increased the fluorescence lifetime compared to control levels. In contrast, zinc oxide decreased the fluorescence lifetime, representing decreased membrane order. However, treatment with soluble zinc sulfate resulted in no significant change in fluorescence lifetime, indicating that the decrease in order of the RBC membranes caused by zinc oxide ENM was not due to zinc ions formed during potential dissolution of the nanoparticles. These results give insight into mechanisms for how these three materials might disrupt RBC membranes and membranes of other cells. The results also provide evidence for a direct correlation between the size, interaction-available surface area of the nano-material and cell membrane disruption.

The Human Cytochrome c Domain-Swapped Dimer Facilitates Tight Regulation of Intrinsic Apoptosis.

Oxidation of cardiolipin (CL) by cytochrome c (cytc) has been proposed to initiate the intrinsic pathway of apoptosis. Domain-swapped dimer (DSD) conformations of cytc have been reported both by our laboratory and by others. The DSD is an alternate conformer of cytc that could oxygenate CL early in apoptosis. We demonstrate here that the cytc DSD has a set of properties that would provide tighter regulation of the intrinsic pathway. We show that the human DSD is kinetically more stable than horse and yeast DSDs. Circular dichroism data indicate that the DSD has a less asymmetric heme environment, similar to that seen when the monomeric protein binds to CL vesicles at high lipid:protein ratios. The dimer undergoes the alkaline conformational transition near pH 7.0, 2.5 pH units lower than that of the monomer. Data from fluorescence correlation spectroscopy and fluorescence anisotropy suggest that the alkaline transition of the DSD may act as a switch from a high affinity for CL nanodiscs at pH 7.4 to a much lower affinity at pH 8.0. Additionally, the peroxidase activity of the human DSD increases 7-fold compared to that of the monomer at pH 7 and 8, but by 14-fold at pH 6 when mixed Met80/H2O ligation replaces the lysine ligation of the alkaline state. We also present data that indicate that cytc binding shows a cooperative effect as the concentration of cytc is increased. The DSD appears to have evolved into a pH-inducible switch that provides a means to control activation of apoptosis near pH 7.0.

Effects of titanium dioxide and zinc oxide nano-materials on lipid order in model membranes.

Engineered nano-materials (ENM) have been reported to affect lipid membrane permeability in cell models, but a mechanistic understanding of how these materials interact with biological membranes has not been described. To assess mechanisms of permeability, liposomes composed of DOPC, DOPS, or POPC, with or without cholesterol, were used as model membranes for measuring ENM-induced changes to lipid order to improve our understanding of ENM effects on membrane permeability. Liposomes were treated with either titanium dioxide (TiO2) or zinc oxide (ZnO) ENM, and changes to lipid order were measured by time-resolved fluorescence anisotropy of a lipophilic probe, Di-4-ANEPPDHQ. Both ENM increased lipid order in two lipid models differing in headgroup charge. TiO2 increased lipid order of POPC liposomes (neutral charge), while ZnO acted primarily on DOPS liposomes (negative charge). Addition of cholesterol to these models significantly increased lipid order while in some cases attenuated ENM-induced changes to lipid order. To assess the ability of ENM to induce membrane permeability, liposomes composed of the above lipids were assayed for membrane permeability by calcein leakage in response to ENM. Both ENM caused a dose-dependent increase in permeability in all liposome models tested, and the addition of cholesterol to the liposome models neither blocked nor reduced calcein leakage. Together, these experiments show that ENM increased permeability of small molecules (calcein) from model liposomes, and that the magnitude of the effect of ENM on lipid order depended on ENM surface charge, lipid head group charge and the presence of cholesterol in the membrane.

Using Time-Resolved Fluorescence Anisotropy of di-4-ANEPPDHQ and F2N12S to Analyze Lipid Packing Dynamics in Model Systems.

The fluorescence probes di-4-ANEPPDHQ and F2N12S have solvochromatic emission spectra and fluorescence lifetimes that are sensitive to order within the environment of lipid membranes. We show in this communication that the time-resolved fluorescence anisotropy of these probes, analyzed either by the wobble-in-a-cone model or by the model-independent order parameter S2, provides complementary information about dynamics and lipid packing in a variety of homogeneous lipid membranes systems.

Trans Influence and Substituent Effects on the HOMO-LUMO Energy Gap and Stokes Shift in Ru Mono-Diimine Derivatives.

The ground (S0) and excited triplet (T1) electronic states and corresponding optical spectra of a series of cationic complexes [RuH(CO)L(PPh3)2]+ (L=2,2´-bipyridyl) (Rubpy), 4,4´-dicarboxylic-2,2´-bipyridyl (Rudcbpy), bis-4,4'-(N-methylamide)-2,2´-bipyridyl (Rudamidebpy), bis-4,4'-(methyl)-2,2´-bipyridyl (RudMebpy), [Ru(CO)2dcbpy(PPh3)2]2+ (Ru(2CO)dcbpy), and [Ru(H)2dcbpy(PPh3)2] (Ru(2H)dcbpy) have been studied by combined Density Functional/Time-Dependent Density Functional (DFT/TDDFT) techniques using different combinations of DFT exchange-correlation functionals and basis sets. PBE0/LANL2DZ provided more accurate geometries to describe S0 whereas B3LYP/LANL2DZ predicted spectral energies that correlated better with the available experiment data. The Ru (II) complexes with different substituents emit photons ranging from 560-610 nm in the series RudMebpy, Rubpy, Rudamidebpy, Rudcbpy. The calculations predicted a maximum emission at about 540 nm for the complex constructed from two carbonyl π-acceptors ligands trans to the dcbpy, while an emission in the far infrared region is calculated when two H σ-donor ligands trans to the dcbpy. Our calculation results show correlations between HOMO-LUMO energy gap, Stokes shift, and T1 distortion, which reflect the different effects of electron-withdrawing and donating groups. We proposed that these correlations can be used to predict the photophysical properties for new complexes.

Synthesis, structure, photophysical and electrochemical properties of Ru(TFA)(CO)(PPh3)2(L) (L=2-phenylpyridine, 2-p-tolylpyridine) and Ru(CO)(PPhMe2)2(L)(L') (L= TFA, H) (L'= bipyridine, L'= 4,4'-dimethylbipyridine) relationships between ancillary ligand structure and luminescent properties.

The synthesis, structure and photophysical properties of the complexes [Ru[(CO)(TFA) (PPh3)2(L)] [(L = ppy = 2-phenylpyridine, (1a); L = 2-(p-tolyl)pyridine] (1b), are reported. The complexes were characterized by UV-VIS, IR and NMR and by single-crystal X-ray diffraction techniques. We also report the synthesis, structure and photophysical properties of [Ru(CO)(L)(PPhMe2)2(L')]+[PF6]- [L' = bipyridine, L = TFA, (3a); L = H, (3b) and L = H, L' = 4,4'-dimethlyl bipyridine (3c)]. These compounds were characterized by UV-VIS, IR and NMR techniques and by a single crystal X-ray diffraction in the case of 3a. The solid state structure of [Ru(Me2PhP)2(CO)2(TFA)2 (2) which is the starting material for the synthesis 3a-3c is also reported to verify the trans relationship of the less bulky PPhMe2 and for comparison with the previously reported PPh3 analogs. The purpose of this study was to determine the impact, if any, of replacing bpy with ppy in the case of 1a and alkylation of the benzene ring in the case of 1b on the photophysical and electrochemical properties compared to related Ru(bpy) complexes. In contrast to the bpy analogs 1a and 1b showed reversible 1e- oxidations and blue-shifted MLCT absorptions. In the case of 3a-3c we were interested in the effect on the photophysical properties of substituting PPh3 with the less bulky but more electron donating PPhMe2. There were only minor changes in the photophysical and electrochemical properties relative to the previously reported PPh3 analogs.

Phospholipid bilayer affinities and solvation characteristics by electrokinetic chromatography with a nanodisc pseudostationary phase.

Phospholipid bilayer nanodiscs composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and synthetic maleic acid-styrene copolymer belts have been introduced as a pseudostationary phase (PSP) in electrokinetic chromatography and demonstrated good performance. The nanodiscs provide a suitable migration range and high theoretical plate counts. Using this nanodisc pseudostationary phase, the affinity of the bilayer structure for probe solutes was determined and characterized. Good correlation is observed between retention factors and octanol water partition coefficients for particular categories of solutes, but the general correlation is weak primarily because the nanodiscs show stronger affinity than octanol for hydrogen bond donors. This suggests that a more appropriate application of this technology is to measure and characterize interactions between solutes and lipid bilayers directly. Linear solvation energy relationship analysis of the nanodisc-solute interactions in this study demonstrates that the nanodiscs provide a solvation environment with low cohesivity and weak hydrogen bond donating ability, and provide relatively strong hydrogen bond acceptor strength.

Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons.

Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate. We present three structures at 1.7-2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and ω-undecylenyl-ß-d-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6-4.0 Å from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.

The Charge Properties of Phospholipid Nanodiscs.

Phospholipids (PLs) are a major, diverse constituent of cell membranes. PL diversity arises from the nature of the fatty acid chains, as well as the headgroup structure. The headgroup charge is thought to contribute to both the strength and specificity of protein-membrane interactions. Because it has been difficult to measure membrane charge, ascertaining the role charge plays in these interactions has been challenging. Presented here are charge measurements on lipid Nanodiscs at 20°C in 100 mM NaCl, 50 mM Tris, at pH 7.4. Values are also reported for measurements made in the presence of Ca(2+) and Mg(2+) as a function of NaCl concentration, pH, and temperature, and in solvents containing other types of cations and anions. Measurements were made for neutral (phosphatidylcholine and phosphatidylethanolamine) and anionic (phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylinositol 4,5-bisphosphate (PIP2)) PLs containing palmitoyl-oleoyl and dimyristoyl fatty acid chains. In addition, charge measurements were made on Nanodiscs containing an Escherichia coli lipid extract. The data collected reveal that 1) POPE is anionic and not neutral at pH 7.4; 2) high-anionic-content Nanodiscs exhibit polyelectrolyte behavior; 3) 3 mM Ca(2+) neutralizes a constant fraction of the charge, but not a constant amount of charge, for POPS and POPC Nanodiscs; 4) in contrast to some previous work, POPC only interacts weakly with Ca(2+); 5) divalent cations interact with lipids in a lipid- and ion-specific manner for POPA and PIP2 lipids; and 6) the monovalent anion type has little influence on the lipid charge. These results should help eliminate inconsistencies among data obtained using different techniques, membrane systems, and experimental conditions, and they provide foundational data for developing an accurate view of membranes and membrane-protein interactions.

Nanosecond Dynamics of Gαi1 Bound to Nucleotides or Ric-8A, a Gα Chaperone with GEF Activity.

Resistance to Inhibitors of Cholinesterase A (Ric-8A) is a 60-kDa cytosolic protein that has chaperone and guanine nucleotide exchange (GEF) activity toward heterotrimeric G protein α subunits of the i, q, and 12/13 classes, catalyzing the release of GDP from Gα and subsequent binding of GTP. In the absence of GTP or GTP analogs, and subsequent to GDP release, Gα forms a stable nucleotide-free complex with Ric-8A. In this study, time-resolved fluorescence anisotropy measurements were employed to detect local motions of Gαi1 labeled at selected sites with Alexa 488 (C5) fluorescent dye (Ax) in the GDP, GTPγS (collectively, GXP), and Ric-8A-bound states. Sites selected for Alexa 488 (C5) derivatization were in the α-helical domain (residue 106), the α-helical domain-Ras-like domain hinge (residue 63), Switch I (residue 180), Switch II (residue 209), Switch III (residue 238), the α4 helix (residue 305), and at the junction between the purine-binding subsite in the ß6-α5 loop and the C-terminal α helix (residue 330). In the GXP-bound states, the Alexa fluorophore reports local motions with correlation times ranging from 1.0 to 1.8 ns. The dynamics at Ax180 is slower in Gαi1•GDP than in Gαi1•GTPγS. The reverse is true at Ax209. The order parameters, S(2), for Alexa probes at switch residues are high (0.78-0.88) in Gαi1•GDP and lower (0.67-0.75) in Gαi1•GTPγS, although in crystal structures, switch segments are more ordered in the latter. Local motions at Ax63, Ax180, Ax209, and Ax330 are all markedly slower (2.3-2.8 ns) in Gαi1:Ric-8A than in Gαi1•GXP, and only modest (± 0.1) differences in S(2) are observed at most sites in Gαi1:Ric-8A relative to Gαi1•GXP. The slow dynamics suggests long-range correlated transitions within an ensemble of states and, particularly in the hinge and switch segments that make direct contact with Ric-8A. Induction of Gαi1 structural heterogeneity by Ric-8A provides a mechanism for nucleotide release.

Luminescence Quenching and Enhancement by Adsorbed Metal Ions with Ruthenium Diimine Complexes Immobilized on Silica Polyamine Composites.

Luminescent ruthenium diimine complexes have been covalently bound to the surface of a silica polyamine composite (SPC) using peptide coupling agents. The loading of the complexes using this route is quite low (~0.01-0.04 mmol/g) leaving sufficient surface amines to coordinate added metal ions. When the composite particles containing the Ru complexes are exposed to solutions of Cu2+, Ni2+ or Zn2+, luminescence is quenched with efficiencies that follow concentration dependence and the relative binding affinities of the ions. When heavy metal ions such as mercury or lead are adsorbed onto the same surface, luminescence is enhanced by a factor of ~3.5. When the complexes are exposed to these metals in solution, no quenching or enhancement is observed. Both phenomena were shown to be the result of adsorption of the cations onto the polyamine surface by using the Stern-Volmer relationship. The mechanism of both quenching and enhancement is discussed and the options for further development of this novel metal sensing technique are presented.

Structural and functional characterization of the acidic region from the RIZ tumor suppressor.

RIZ (retinoblastoma protein-interacting zinc finger protein), also denoted PRDM2, is a transcriptional regulator and tumor suppressor. It was initially identified because of its ability to interact with another well-established tumor suppressor, the retinoblastoma protein (Rb). A short motif, IRCDE, in the acidic region (AR) of RIZ was reported to play an important role in the interaction with the pocket domain of Rb. The IRCDE motif is similar to a consensus Rb-binding sequence LXCXE (where X denotes any amino acid) that is found in several viral Rb-inactivating oncoproteins. To improve our understanding of the molecular basis of binding of Rb to RIZ, we investigated the interaction between purified recombinant AR and the pocket domain of Rb using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and fluorescence anisotropy experiments. We show that AR is intrinsically disordered and that it binds the pocket domain with submicromolar affinity. We also demonstrate that the interaction between AR and the pocket domain is mediated primarily by the short stretch of residues containing the IRCDE motif and that the contribution of other parts of AR to the interaction with the pocket domain is minimal. Overall, our data provide clear evidence that RIZ is one of the few cellular proteins that can interact directly with the LXCXE-binding cleft on Rb.

Surface-Bound Ruthenium Diimine Organometallic Complexes: Excited-State Properties.

Ruthenium complexes of the general formula [Ru(CO)(H)(L2)(L'2)][PF6] (L2 = trans-2PPh3, L' = η2-4,4'-dicarboxybipyridine (1); L2 =trans-2Ph2PCH2CH2COOH, L'2 = bipyridine (2); L2 = Ph2PCHCHPPh2, L' = η2-5-amino-1,10-phenanthroline (3); L2 = trans-2PPh3, L'2 = η2-4-carboxaldehyde-4'-methylbipyridine (4)) have been shown to have longer emission lifetimes and higher quantum yields in solution compared with more symmetrical molecules such as [Ru(bpy)3][Cl]2. Compound 4 is obtained as a mixture with the corresponding acetal, 4'. These less symmetrical complexes have been covalently immobilized on the surface of silica polyamine composites, and their photophysical properties have been studied. The surface-bound complexes have been characterized by solid-state CPMAS 13C, 31P, and 29Si NMR, UV-vis, and FT-IR spectroscopies. Excited-state lifetime studies revealed that, in general, the lifetimes of the immobilized complexes are 1.4 to 8 times longer than in solution and are dependent on particle size (300-500 µm versus 10-20 nm average diameter silica gels), polymer structure (linear poly(allylamine) versus branched poly(ethylenimine)), and the type of surface tether. One exception to this trend is the previously reported complex [Ru(bpy)2(5-amino-1,10-phenanthroline)][PF6]2 (5), where only a slight increase in lifetime is observed. Only minor changes in emission wavelength are observed for all the complexes. This opens up the possibility for enhanced heterogeneous electron transfer in photocatalytic reactions.

Photophysical studies of bioconjugated ruthenium metal-ligand complexes incorporated in phospholipid membrane bilayers.

The luminescent, mono-diimine ruthenium complexes [(H)Ru(CO)(PPh3)2(dcbpy)][PF6] (1) (dcbpy = 4,4'-dicarboxy-2,2'-bipyridyl) and [(H)Ru(CO)(dppene)(5-amino-1,10-phen)][PF6] (2) (dppene = bis(diphenylphosphino)ethylene; phen = phenanthroline) were conjugated with 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE) and with cholesterol in the case of complex 2. Using standard conjugation techniques, compound 1 gives the bis-lipid derivative [(H)Ru(CO)(PPh3)2(dcbpy-N-DPPE2)][PF6] (3), while 2 provides the monolipid conjugate [(H)Ru(CO)(dppene)(1,10-phen-5-NHC(S)-N-DPPE)][PF6] (4) and the cholesterol derivative [(H)Ru(CO)(dppene)(1,10-phen-5-NHC(O)Ocholesteryl)][PF6] (5). These compounds were characterized by spectroscopic methods, and their photophysical properties were measured in organic solvents. The luminescence of lipid conjugates 3 and 4 is quenched in organic solvents while compound 4 shows a weak, short-lived, blue-shifted emission in aqueous solution. The cholesterol conjugate 5 shows the long-lived, microsecond-time scale emission associated with triplet metal-to-ligand charge-transfer excited states. Incorporation of conjugate 3 in lipid bilayer vesicles restores the luminescence, but with blue shifts (~80 nm) accompanied by nanosecond-time scale lifetimes. In the vesicles conjugate 4 shows a short-lived and blue-shifted emission similar to that observed in solution but with increased intensity. Conjugation of the complex [(H)Ru(CO)(PhP2C2H4C(O)O-N-succinimidyl)2(bpy)][PF6] (6") (bpy = 2,2'-bipyridyl) with DPPE gives the phosphine-conjugated complex [(H)Ru(CO)(PhP2C2H4C(O)-N-DPPE)2(bpy)][PF6] (7). Complex 7 also exhibits a short-lived and blue-shifted emission in solution and in vesicles as observed for complexes 3 and 4. We have also conjugated the complex [Ru(bpy)2(5-amino-1,10-phen)][PF6]2 (8) with both cholesterol (9) and DPPE (10). Neither complex 9 nor the previously reported complex 10 exhibited the blue shifts observed for complexes 3 and 4 when incorporated into large unilamellar vesicles (LUVs). The anisotropies of the emissions of complexes 3, 4, and 7 were also measured in LUVs, and those of complex 5 were measured in both glycerol and LUVs. High fundamental anisotropies were observed for complexes 3, 4, and 7.

One-step Melt Synthesis of Water Soluble, Photoluminescent, Surface-Oxidized Silicon Nanoparticles for Cellular Imaging Applications.

We have developed a versatile, one-step melt synthesis of water-soluble, highly emissive silicon nanoparticles using bi-functional, low-melting solids (such as glutaric acid) as reaction media. Characterization through transmission electron microscopy, selected area electron diffraction, X-ray photoelectron spectroscopy, and Raman spectroscopy shows that the one-step melt synthesis produces nanoscale Si cores surrounded by a silicon oxide shell. Analysis of the nanoparticle surface using FT-IR, zeta potential, and gel electrophoresis indicates that the bi-functional ligand used in the one-step synthesis is grafted onto the nanoparticle, which allows for tuning of the particle surface charge, solubility, and functionality. Photoluminescence spectra of the as-prepared glutaric acid-synthesized silicon nanoparticles show an intense blue-green emission with a short (ns) lifetime suitable for biological imaging. These nanoparticles are found to be stable in biological media and have been used to examine cellular uptake and distribution in live N2a cells.

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway.

Localized DNA melting may provide a general strategy for recognition of the wide array of chemically and structurally diverse DNA lesions repaired by the nucleotide excision repair (NER) pathway. However, it is not clear what causes such DNA melting and how it is driven. Here, we show a DNA wrapping-melting model supported by results from dynamic monitoring of the key DNA-protein and protein-protein interactions involved in the early stages of the Escherichia coli NER process. Using an analytical technique involving capillary electrophoresis coupled with laser-induced fluorescence polarization, which combines a mobility shift assay with conformational analysis, we demonstrate that DNA wrapping around UvrB, mediated by UvrA, is an early event in the damage-recognition process during E. coli NER. DNA wrapping of UvrB was confirmed by Förster resonance energy transfer and fluorescence lifetime measurements. This wrapping did not occur with readily denaturable damaged DNA substrates ("bubble" DNA), suggesting that DNA wrapping of UvrB plays an important role in the induction of DNA melting around the damage site. Analysis of DNA wrapping of mutant UvrB Y96A further suggests that a cooperative interaction between DNA wrapping of UvrA(2)B and contact of the beta-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site.

Loop dynamics of the extracellular domain of human tissue factor and activation of factor VIIa.

In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro(79)-Pro(92) loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu(84) resides in the Pro(79)-Pro(92) loop, and Thr(121) resides in the turn between the first and second antiparallel beta-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu(84) nor Thr(121) makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro(79)-Pro(92) loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro(79)-Pro(92) loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation.

Tuning photophysical properties with ancillary ligands in Ru(II) mono-diimine complexes.

The series of complexes [XRu(CO)(L-L)(L')2][PF6] (X = H, TFA, Cl; L-L = 2,2'-bipyridyl, 1,10-phenanthroline, 5-amino-1,10-phenanthroline and 4,4'-dicarboxylic-2,2'-bipyridyl; L'2 = 2PPh3, Ph2 PC2H4PPh2, Ph2PCH═CHPPh2) have been synthesized from the starting complex K[Ru(CO)3(TFA)3] (TFA = CF3CO2) by first reacting with the phosphine ligand, followed by reaction with the L-L and anion exchange with NaPF6. In the case of L-L = phenanthroline and L'2 = 2PPh3, the neutral complex Ru(Ph3P)(CO)(1,10-phenanthroline)( TFA)2 is also obtained and its solid state structure is reported. Solid state structures are also reported for the cationic complexes where L-L = phenanthroline, L2 = 2PPh3 and X = Cl and for L-L = 2,2'-bipyridyl, L2 = 2PPh3 and X = H. All the complexes were characterized in solution by a combination of 1H and 31P NMR, IR, mass spectrometry and elemental analyses. The purpose of the project was to synthesize a series of complexes that exhibit a range of excited-state lifetimes and that have large Stokes shifts, high quantum yields and high intrinsic polarizations associated with their metal-to-ligand charge-transfer (MLCT) emissions. To a large degree these goals have been realized in that excited-state lifetimes in the range of 100 ns to over 1 µs are observed. The lifetimes are sensitive to both solvent and the presence of oxygen. The measured quantum yields and intrinsic anisotropies are higher than for previously reported Ru(II) complexes. Interestingly, the neutral complex with one phosphine ligand shows no MLCT emission. Under the conditions of synthesis some of the initially formed complexes with X = TFA are converted to the corresponding hydrides or in the presence of chlorinated solvents to the corresponding chlorides, testifying to the lability of the TFA Ligand. The compounds show multiple reduction potentials which are chemically and electrochemically reversible in a few cases as examined by cyclic voltammetry. The relationships between the observed photophysical properties of the complexes and the nature of the ligands on the Ru(II) is discussed.

Synthesis, structure, photophysical and electrochemical behavior of 2-amino-anthracene triosmium clusters.

The reactions of 2-amino-anthracene with [Os3(CO)10(CH3CN)2] have been studied and the products structurally characterized by spectroscopic, X-ray diffraction, photophysical and electrochemical techniques. At room temperature in CH2Cl2 two major, isomeric products are obtained [Os3(CO)10(µ-η2-(N-C(1))-NH2C14H8)(µ-H)] (1, 14%) and [Os3(CO)10(µ-η2-(N-C(3))-NHC14H9)(µ-H)] (2, 35%) along with a trace amount of the dihydrido complex [Os3(CO)9(µ-η2-(N-C(3))-NHC14H8)(µ-H)2] (3). In refluxing tetrahydrofuran only complexes 2 and 3 are obtained in 24% and 28%, respectively. A separate experiment shows that complex 1 slowly converts to 2 and that the rearrangement is catalyzed by adventitious water and involves proton transfer to the anthracene ring. Complex 1 is stereochemically non-rigid; exhibiting edge to edge hydride migration while 2 is stereochemically rigid. Complex 3 is also stereochemically non-rigid showing a site exchange process of the magnetically nonequivalent hydrides typical for trinuclear dihydrides. Interestingly, 2 decarbonylates cleanly to the electronically unsaturated 46e- cluster [Os3(CO)9(µ3-η2-(N-C(3))-NHC10H9)(µ-H)] (4, 68%) in refluxing cyclohexane, while photolysis of 2 in CH2Cl2 yields only a small amount of 3 along with considerable decomposition. The mechanism of the conversion of 1 to 2 and the dependence of the product distribution on solvent are discussed. All four compounds are luminescent with compounds 1-3 showing emissions that can be assigned to radiative decay associated with the anthracene ligand. Complexes 1-3 all show irreversible 1e- reductions in the range of-1.85-2.14 V while 4 shows a nicely reversible 1e- wave at-1.16 V and a quasi-reversible second 1e- wave at-1.62 V. Irreversible oxidations are observed in the range from +0.35 to +0.49 V. The relationship between the cluster ligand configurations and the observed electrochemical and photochemical behavior is discussed and compared with that of the free ligand.

Spectroscopic and computational studies of a Ru(II) terpyridine complex: the importance of weak intermolecular forces to photophysical properties.

The complex [Ru(tpy)(CO)(2)TFA]+[PF(6)]- (where tpy = 2,2':6',2' '-terpyridine and TFA = CF(3)CO(2)-) (1) has been synthesized and fully characterized spectroscopically. The X-ray structure of the complex has been determined. The photopysical properties of the ruthenium complex and the free ligand tpy have been investigated at room temperature and at 77 K in acetonitrile solution and in the solid state. Their electronic spectra are highly influenced by intermolecular stacking interactions, both in solution and in the solid state. Density functional theory (DFT) and time-dependent DFT (TDDFT) calculations have been performed to characterize the electronic structure and the excited states of [Ru(tpy)(CO)(2)TFA]+[PF(6)]- and tpy. TDDFT calculations on three different conformations of free ligand have been performed as well. Absorption and emission spectra of tpy have been studied at different temperatures and concentrations in order to have a better understanding of this ruthenium derivative's properties. The absorption spectrum of 1 is characterized by metal-perturbed ligand-centered (LC) bands in the UV region. No metal-to-ligand charge transfer (MLCT) bands are observed in the visible for the complex. Only at high concentrations (10(-4) M) does a very weak band appear at 470 nm. At 77 K and low concentrations, solutions of 1 exhibit a major 3LC emission band centered at 468 nm (21.4 x 10(-3) cm(-1)). When the concentration of the complex is increased, an unstructured narrow emission at 603 nm (16.6 x 10(-3) cm(-1)), with a lifetime of 10 micros, dominates the emission spectrum in glassy acetonitrile. This emission originates from a pi-pi stacked dimeric (or oligomeric) species. TDDFT calculations performed on a tail-to-tail dimer structure, similar to that seen in the solid state, ascribe the transition to a triplet excited state, where intermolecular metal (d) --> ligand (pi*, polypyridine) charge transfer occurs. A good estimate of the transition energy is also obtained (623 nm, 1.94 eV).