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Quenching Constant (quenching + constant)
Selected AbstractsFlexibility in Proteins: Tuning the Sensitivity to O2 Diffusion by Varying the Lifetime of a Phosphorescent Sensor in Horseradish Peroxidase,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2004Janna Nibbs ABSTRACT The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656,666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. Kd= 18 ,M when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O2 to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of kq= 234 mm Hg,1 s,1 in the absence of BHA and kq= 28.7 mm Hg,1 s,1 in the presence of BHA. The quenching rate of BHA is 4000 s,1. The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O2 diffusion and flexibility in heme proteins. [source] Molecular interactions of isoxazolcurcumin with human serum albumin: Spectroscopic and molecular modeling studiesBIOPOLYMERS, Issue 2 2009Bijaya Ketan Sahoo Abstract Curcumin is a nontoxic natural product with diverse pharmacological potencies. We report the interaction of a potent synthetic derivative of curcumin, isoxazolcurcumin (IOC) with human serum albumin (HSA) using various biophysical methods. The observed fluorescence quenching of HSA by IOC is due to a complex formation by a static quenching process with a quenching constant of the order of 105M,1. The binding affinity and the number of binding sites were obtained from a Scatchard analysis. Thermodynamics reveals that the interaction is entropy driven with predominantly hydrophobic forces. From the observed Förster-type fluorescence resonance energy transfer (FRET), the donor (Trp 214 in HSA) to acceptor (IOC) distance is calculated to be 3.2 nm. The conformational changes of HSA due to the interaction were investigated qualitatively from synchronous fluorescence spectra along with a quantitative estimation of the secondary structure from Fourier Transform Infrared (FTIR) and circular dichroism (CD) spectroscopies. Molecular docking studies were performed to obtain information on the possible residues involved in the interaction process, and changes in accessible surface area of the interacting residues were calculated. The preferred binding site of IOC was analyzed by ligand displacement experiments with 1-anilino-8-naphthalenesulfonate (ANS) and warfarin-bound HSA. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 108,119, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Spectroscopic studies of interaction of chlorobenzylidine with DNABIOPOLYMERS, Issue 6 2001Wenying Zhong Abstract Electronic absorbance and fluorescence titrations are used to probe the interaction of chlorobenzylidine with DNA. The binding of chlorobenzylidine to DNA results in hypochromism, a small shift to a longer wavelength in the absorption spectra, and emission quenching in the fluorescence spectra. These spectral characteristics suggest that chlorobenzylidine binds to DNA by an intercalative mode. This conclusion is reinforced by fluorescence polarization measurements. Scatchard plots constructed from fluorescence titration data give a binding constant of 1.3 × 105M,1 and a binding site size of 10 base pairs. This indicates that chlorobenzylidine has a high affinity with DNA. The intercalative interaction is exothermic with a Van't Hoff enthalpy of ,143 kJ/mol. This result is obtained from the temperature dependence of the binding constant. The interaction of chlorobenzylidine with DNA is affected by the pH value of the solution. The binding constant has its maximum at pH 3.0. Upon binding to DNA, the fluorescence from chlorobenzylidine is quenched efficiently by the DNA bases and the fluorescence intensity tends to be constant at high concentrations of DNA when the binding is saturated. The Stern,Volmer quenching constant obtained from the linear quenching plot is 1.6 × 104M,1 at 25°C. The measurements of the fluorescence lifetime and the dependence of the quenching constant on the temperature indicate that the fluorescence quenching process is static. The fluorescence lifetime of chlorobenzylidine is 1.9 ± 0.4 ns. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 315,323, 2001 [source] Synthesis, Characterization, and Application of Cationic Water-Soluble Oligofluorenes in DNA-Hybridization DetectionCHEMISTRY - A EUROPEAN JOURNAL, Issue 24 2008Bin Liu Prof. Abstract A simple and efficient approach was developed for the synthesis of a series of cationic water-soluble oligofluorenes up to a chain length of a heptamer. Bromoalkyl-substituted fluorenyl boronic esters as the key intermediates were synthesized by using a modified Miyaura reaction. With an increasing number of repeat units (trimer to hexamer), the size-specific oligomers have shown redshifts in both the absorption and emission maxima. The emission maximum reaches the limit for the hexamer in both water and buffer solution. The quantum yields of the oligomers decreased with increased oligomer size in water. Both fluorescence quenching of the oligomers by 9,10-anthraquinone-2,6-disulfonate and the fluorescence resonance energy transfer experiments with the oligomers as the donor and fluorescein (Fl)-labeled double-stranded DNA (dsDNA-Fl) as the acceptor revealed the chain-length-dependent behavior. The Stern,Volmer quenching constant increased with the molecular size, whereas the highest donor-sensitized Fl emission was observed for the hexamer. These size-specific oligomers also served as a model to study the structure,property relationships for cationic polyfluorenes. [source] A Close Look at Fluorescence Quenching of Organic Dyes by TryptophanCHEMPHYSCHEM, Issue 11 2005Sören Doose Dr. Abstract Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant kdof 4.0×109s,1,M,1, 2) static quenching with a bimolecular association constant Ksof 61,M,1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant , of 22,M,1. The latter two are characterized as nonfluorescent complexes, formed with ,30,% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 20,30 kJmol,1is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales. [source] Excited Singlet (S1)-state Interactions of Nile Red with Aromatic Amines,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2003J. Mohanty ABSTRACT Both steady-state (SS) and time-resolved (TR) studies show that the fluorescence of the dye Nile red (NR) is quenched by various aromatic amines (ArA). Bimolecular quenching constants (kq) from both SS and TR measurements are observed to match well, indicating that the interaction is dynamic in nature. The quenching interaction in the present systems has been attributed to electron transfer (ET) from ArA to excited NR, based on the variations in the kq values with the oxidation potentials of the amines. The kq values calculated within the framework of Marcus' outer-sphere ET theory at different free-energy changes (,G0) of the ET reactions match well with the experimental ones, supporting the ET mechanism in the systems studied. The reorganization energy (,) estimated from the correlation of the experimental and the calculated kq values is quite similar to the solvent reorganization energy (,s), calculated on the basis of the solvent dielectric continuum model along with the assumption that the reactants are the effective spheres. Although a modest error is involved in this ,s calculation, the similarity in , and ,s values suggests that the solvent reorganization plays a dominant role in governing the ET dynamics in the present systems. [source] The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrinPROTEIN SCIENCE, Issue 9 2009Yuanli Song Abstract The bundling of the N-terminal, partial domain helix (Helix C,) of human erythroid ,-spectrin (,I) with the C-terminal, partial domain helices (Helices A, and B,) of erythroid ,-spectrin (,I) to give a spectrin pseudo structural domain (triple helical bundle A,B,C,) has long been recognized as a crucial step in forming functional spectrin tetramers in erythrocytes. We have used apparent polarity and Stern,Volmer quenching constants of Helix C, of ,I bound to Helices A, and B, of ,I, along with previous NMR and EPR results, to propose a model for the triple helical bundle. This model was used as the input structure for molecular dynamics simulations for both wild type (WT) and ,I mutant L49F. The simulation output structures show a stable helical bundle for WT, but not for L49F. In WT, four critical interactions were identified: two hydrophobic clusters and two salt bridges. However, in L49F, the region downstream of Helix C, was unable to assume a helical conformation and one critical hydrophobic cluster was disrupted. Other molecular interactions critical to the WT helical bundle were also weakened in L49F, possibly leading to the lower tetramer levels observed in patients with this mutation-induced blood disorder. [source] Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalinPROTEIN SCIENCE, Issue 2 2000Oktay K. Gasymov Abstract The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a ,-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography. [source] | ||||||||||