Search

The ground (S 0 ) and excited triplet (T 1 ) electronic states and corresponding optical spectra of a series of cationic complexes [RuH(CO)L(PPh 3 ) 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) 2 dcbpy(PPh 3 ) 2 ] 2+ (Ru(2CO)dcbpy), and [Ru(H) 2 dcbpy(PPh 3 ) 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 S 0 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 T 1 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.

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., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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 Cu 2+ , Ni 2+ or Zn 2+ , 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.

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236-39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379-401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF-DNA interfaces while the resulting energetic compensation maintains the same net binding energy.

The synthesis and physical-chemical characterization of the metal-ligand complex [Os(bpy)2(CO)(enIA)][OTf]2 (where enIA = ethylenediamine iodoacetamide) with a sulfhydryl-specific functional group is described. The UV and visible absorption and luminescence emission, including lifetime and steady-state anisotropy, are reported for the free probe and the probe covalently linked to two test proteins. The spectroscopic properties of the probe are unaffected by chemical modification and subsequent covalent linkage to the proteins. The luminescence lifetime in aqueous buffer is approximately 200 ns and the limiting anisotropy is greater than 0.125, suggesting a potentially useful probe for biophysical investigations.

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)(n). About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5-9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter ( approximately 3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels.

The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated.

We have measured the steady-state phosphorescence and decay times of wild-type λcI repressor and compared it with that of a modified λcI repressor in which > 95% of the tryptophans were replaced with 5-hydroxy-L-tryptophan (5-OHTrp). The wild-type and 5-OHTrp-λcI repressors are spectroscopically distinct such that we can selectively excite the 5-OHTrp-λcI even in the presence of a 15-fold molar excess ofN-acetyltryptophanamide (NATrpA). The phosphorescence band of wild-type λcI is red-shifted by 3 nm relative to NATrpA, characteristic of buried tryptophan. Similarly, the phosphorescence of 5-OHTrp-λcI repressor is red-shifted relative to the model, 5-OHTrp, showing that according to the phosphorescence, the modified repressor is structurally indistinguishable from the native repressor. While the phosphorescence decay of both NATrpA and 5-OHTrp are single exponentials, the decay of both wild-type and 5-OHTrp-λcI repressors is complex, requiring three decay components whose fractional contributions to the phosphorescence are the same for both repressors. Because the 5-OHTrp phosphorescence can be excited at wavelengths outside the absorbance range of tryptophan and DNA, a protein spectrally enhanced with this emitter will aid the investigations of protein-protein or protein-DNA interactions.