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PubMed2004

Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer.

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greater donor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB is essentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Ca is due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.

Calcium↗
PubMed2002

Pulsed EPR studies of nonexchangeable protons near the Mo(V) center of sulfite oxidase: direct detection of the alpha-proton of the coordinated cysteinyl residue and structural implications for the active site.

Pulsed electron nuclear double resonance (ENDOR) spectra of nonexchangeable protons in the vicinity of the Mo(V) center of the high pH (hpH) and low pH (lpH) forms of native chicken liver sulfite oxidase (SO) and recombinant human SO have been obtained and analyzed for the first time. The close similarity of the spectra for the chicken and human enzymes indicates that the structures of their molybdenum centers are essentially identical. For lpH SO, the closest nonexchangeable proton is found to be approximately 2.8 A from the Mo atom. To more accurately determine the distance to this proton and facilitate its assignment, the C-band electron spin-echo envelope modulation (ESEEM) spectra of lpH SO were also analyzed. From the obtained distance and comparison with the X-ray structure, this closest nonexchangeable proton is assigned to the alpha-proton of the coordinated conserved cysteinyl residue (Cys185 in chicken, Cys207 in human). The closest Mo...H distance for the nonexchangeable protons of hpH SO is found to be approximately 3.3 A. For the cysteinyl alpha-proton, such an increase in the Mo...H distance only requires a very small change in torsional angles. This study demonstrates that details of the enzyme structural rearrangements with pH can be monitored by ENDOR spectroscopy and suggests that a similar approach may be routinely used to probe the orientation of the coordinated cysteinyl residue in mutant forms of SO that are catalytically compromised.

Animals↗
PubMed2002

The direct detection of a hydrogen bond in the solid state by NMR through the observation of a hydrogen-bond mediated (15)N [bond] (15)N J coupling.

A new method for detecting hydrogen bonds in the solid state is presented. Using two-dimensional NMR correlation experiments, it is shown that a hydrogen-bond mediated J coupling can be observed in a powder under magic-angle spinning conditions, even though the J coupling is 2 orders of magnitude smaller than the dominant anisotropic interactions encountered in solid-state NMR. Specifically, the observation of a pair of peaks in a two-dimensional 15N-15N solid-state INADEQUATE experiment due to two nitrogens that have no covalent connectivity is attributed to the presence of a J coupling across a linking hydrogen bond.

Amines↗
PubMed2002

Direct detection of radical cations of NADH analogues.

The radical cation of an NADH analogue (BNAH: 1-benzyl-1,4-dihydronicotinamide) has been successfully detected as the transient absorption and ESR spectra in the thermal electron transfer from BNAH to Fe(bpy)33+ (bpy = 2,2'-bipyridine). The ESR spectra of the radical cations of BNAH and the dideuterated compound (BNAH-4,4'-d2) indicate that the observed radical cation is the keto form rather than the enol form in the tautomerization. The deprotonation rate and the kinetic isotope effects of the keto form of BNAH*+ were determined from the kinetic analysis of the electron-transfer reactions.

Cations↗
PubMed2003

Direct detection of potassium cations bound to G-quadruplex structures by solid-state 39K NMR at 19.6 T.

We report solid-state 39K NMR detection of the K+ ions bound to three G-quadruplex structures formed by self-assembly of 5'-tert-butyl-dimethylsilyl-2',3'-O-isopropylidene guanosine, guanosine, and guanosine 5'-monophosphate. The 39K NMR spectra clearly show different spectral signatures for K+ ions inside the G-quadruplex channel and for K+ ions bound to the phosphate groups. Solid-state 39K NMR spectra for hydrated K salts of adenosine 2'-monophosphate and adenosine 5'-diphosphate are also reported.

Cations, Monovalent↗
PubMed2004

Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase.

The reaction of substrate-bound taurine/alpha-ketoglutarate dioxygenase with O2 has been studied using cryogenic continuous-flow spectroscopy. Transient absorption spectra acquired at -38 degrees C show an exponential decay of a 318-nm chromophore with an apparent rate of 1.3 s-1. The observed optical changes and their kinetics are consistent with the profile of an Fe(IV) species detected recently by Mössbauer spectroscopy (Price et al., Biochemistry 2003, 42, 7497-7508). Resonance Raman measurement upon excitation at 363.7 nm reveal at least two oxygen isotope-sensitive vibrations at 821/787 cm-1 and 583/555 cm-1 for 16O and 18O derivatives, respectively. An additional mode is likely to be obscured by an ethylene glycol vibration at 865 cm-1 and/or 1089 cm-1. The 821 cm-1 vibration is assigned to the stretching mode of Fe(IV)=O species on the basis of its frequency and isotopic shift amplitude. The 583 cm-1 band is likely to originate from an Fe-O2 precursor of the Fe(IV)=O species, although its structural details are unclear at present.

Mixed Function Oxygenases↗
PubMed2004

Direct detection of formate ligation in cytochrome c oxidase by ATR-FTIR spectroscopy.

The IR signature of binding of formate to the heme a(3-)Cu(B) binuclear site of bovine cytochrome c oxidase has been obtained by perfusion ATR-FTIR spectroscopy. The data show unequivocally that formate binds in its anionic form despite its binding being electroneutral overall. The bound formate can be distinguished from free ligand by the binding-induced sharpening and downshifting of vibrational bands. Formate ligation also causes shifts of vibrational modes of heme a(3) and its substituents and perturbation of histidine residues. The association of the accompanying protonation change with a carboxylate or tyrosine can be ruled out and may involve a histidine metal ligand or, more likely, a simple displacement into the bulk phase of a hydroxide ligand to heme a(3) or CU(B), a reaction which would account for stoichiometric proton uptake and maintenance of net charge within the binuclear center domain.

Animals↗
PubMed2005

Direct detection of dimethylstannylene and tetramethyldistannene in solution and the gas phase by laser flash photolysis of 1,1-dimethylstannacyclopent-3-enes.

The photochemistry of 1,1-dimethyl- and 1,1,3,4-tetramethylstannacyclopent-3-ene (4a and 4b, respectively) has been studied in the gas phase and in hexane solution by steady-state and 193-nm laser flash photolysis methods. Photolysis of the two compounds results in the formation of 1,3-butadiene (from 4a) and 2,3-dimethyl-1,3-butadiene (from 4b) as the major products, suggesting that cycloreversion to yield dimethylstannylene (SnMe2) is the main photodecomposition pathway of these molecules. Indeed, the stannylene has been trapped as the Sn-H insertion product upon photolysis of 4a in hexane containing trimethylstannane. Flash photolysis of 4a in the gas phase affords a transient absorbing in the 450-520-nm range that is assigned to SnMe2 by comparison of its spectrum and reactivity to those previously reported from other precursors. Flash photolysis of 4b in hexane solution affords results consistent with the initial formation of SnMe2 (lambda(max) approximately 500 nm), which decays over approximately 10 micros to form tetramethyldistannene (5b; lambda(max) approximately 470 nm). The distannene decays over the next ca. 50 micros to form at least two other longer-lived species, which are assigned to higher SnMe2 oligomers. Time-dependent DFT calculations support the spectral assignments for SnMe2 and Sn2Me4, and calculations examining the variation in bond dissociation energy with substituent (H, Me, and Ph) in disilenes, digermenes, and distannenes rule out the possibility that dimerization of SnMe2 proceeds reversibly. Addition of methanol leads to reversible reaction with SnMe2 to form a transient absorbing at lambda(max) approximately 360 nm, which is assigned to the Lewis acid-base complex between SnMe2 and the alcohol.

Journal Article↗
PubMed2005

The direct detection of an aryl azide excited state: an ultrafast study of the photochemistry of para- and ortho-biphenyl azide.

Ultrafast laser flash photolysis (266 nm) of para- and ortho-biphenyl azide in acetonitrile produces azide excited states that have broad absorption bands centered at 480 nm. The para-biphenyl azide excited singlet state has a lifetime of 100 fs. The excited-state lifetime of the ortho-azide isomer is 450 +/- 150 fs. Decay of the azide excited states is accompanied by the formation of the corresponding known singlet nitrenes (para, lambdamax = 350 nm, ortho, lambdamax = 400 nm). Singlet para-biphenylnitrene is born with excess energy and undergoes vibrational cooling with a time constant of 11 ps to form the long-lived (tau approximately 9 ns) relaxed singlet nitrene. Singlet ortho-biphenylnitrene decays with a lifetime of 16 ps in acetonitrile at ambient temperature.

Azides↗
PubMed2006

Direct-detected 13C NMR to investigate the iron(III) hemophore HasA.

Hemophore HasA is a 19 kDa iron(III) hemoprotein that participates in the shuttling of heme to a specific membrane receptor. In HasA, heme iron has an original coordination environment with a His/Tyr pair as axial ligands. Recently developed two-dimensional protonless (13)C-detected experiments provide the sequence-specific assignment of all but three protein residues in the close proximity of the paramagnetic center, thus overcoming limitations due to the short relaxation times induced by the presence of the iron(III) center. Mono-dimensional (13)C and (15)N experiments tailored for the detection of paramagnetic signals allow the identification of resonances of the axial ligands. These experiments are used to characterize the conformational features and the electronic structure of the heme iron(III) environment. The good complementarity among (1)H-, (13)C-, and (15)N-detected experiments is highlighted. A thermal high-spin/low-spin equilibrium is observed and is related to a modulation of the strength of the coordination bond between the iron and the Tyr74 axial ligand. The key role of a neighboring residue, His82, for the stability of the axial coordination and its involvement in the heme delivery to the receptor is discussed.

Animals↗
PubMed2003

Photolysis of alpha-azidoacetophenones: direct detection of triplet alkyl nitrenes in solution.

We report the first detection of triplet alkyl nitrenes in fluid solution by laser flash photolysis of alpha-azido acetophenone derivatives, 1. Alphazides 1 contain an intramolecular triplet sensitizer, which ensures formation of the triplet alkyl nitrene by bypassing the singlet nitrene intermediate. At room temperature, azides 1 cleave to form benzoyl and methyl azide radicals in competition with triplet energy transfer to form triplet alkyl nitrene. The major photoproduct 3 arises from interception of the triplet alkyl nitrene with benzoyl radicals. The triplet alkyl nitrene intermediates are also trapped with molecular oxygen to yield the corresponding 2-nitrophenylethanone. Laser flash photolysis of 1 reveals that the triplet alkyl nitrenes have absorption around 300 nm. The triplet alkyl nitrenes were further characterized by obtaining their UV and IR spectra in argon matrices. (13)C and (15)N isotope labeling studies allowed us to characterize the C-N stretch of the nitrene intermediate at 1201 cm(-)(1).

Journal Article↗
PubMed2006

Direct detection of OH radicals diffused to the gas phase from the UV-irradiated photocatalytic TiO2 surfaces by means of laser-induced fluorescence spectroscopy.

The important roles of OH radicals for remote oxidation using TiO(2) photocatalysts were evidenced by the in situ detection of OH radicals in the gas phase using the laser-induced fluorescence (LIF) technique. The appearance of OD-LIF intensities after the exposure of D(2)O vapors over TiO(2) powders and the decrease of the time-resolved signals of OH-LIF intensities with increasing calcined temperatures of TiO(2) powders suggested that the exchangeable water at the TiO(2) surface is the origin of the diffused OH radicals.

Letter↗
PubMed2005

Amperometric immunosensor for direct detection based upon functional lipid vesicles immobilized on nanowell array electrode.

An original electrochemical immunosensor has now been developed that is based upon the spontaneous immobilization of biotinylated, functional lipid vesicles (FLVs) on a polymeric resist layer. An electrode was fabricated utilizing a form of electron-beam (e-beam) that has been used to fabricate 200 nm (nanoscale) wells in the resist layer covering of the gold electrode. The stability of adhered FLVs upon the nanowell (NW) electrode was observed by atomic force microscopy (AFM). From these observations, we were able to determine that the assembled FLVs primarily adhered as individual molecules, that is, without the aggregation or fusion noted in earlier designs. Additionally, these immobilized FLVs demonstrated clearly defined redox activity in electrochemical measurements. Streptavidin, biotinylated capture antibody, and target proteins were consequently injected in order to set up the immunoassay environment. Electrochemical immunoassay experimentation was performed on the NW array electrode with model proteins, such as human serum albumin (HSA) and carbonic anhydrase from bovine (CAB). We observed a notable current decrease, following the redox path, interrupted by the target HSA, indicating the binding of the capture antibody with the target antigen. On the basis of these results, we propose a new type of immunosensor incorporating this mechanism of spontaneous immobilization of FLVs.

Electrochemistry↗
PubMed2003

Direct detection of ion pair formation and collapse in a migration reaction of a beta-phosphate radical.

In solutions of trifluorotoluene or toluene containing 2,2,2-trifluoroethanol, the beta-phosphate radical heterolyzed to give a detectable ion pair, identified as a solvent-separated species. Rate constants for the radical fragmentation reaction forming the ion pair, for ion pair collapse, and for diffusive escape to free ions were measured. The kinetics and entropy of activation for fragmentation indicate that the rearrangement reaction occurs by a heterolysis pathway in all solvents. [reaction: see text]

Journal Article↗
PubMed2002

Evaluation of the Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium bovis in bovine tissue samples.

The commercial LCx amplification assay, usually employed to detect the Mycobacterium tuberculosis complex in respiratory specimens, was evaluated by comparing the results it gave with those obtained using Lowenstein-Jensen solid medium and pathological findings in 55 lymph nodes from cattle with positive and 10 lymph nodes from cattle with negative skin tests for tuberculosis. Fifty-three cultures (51 and 2, respectively) were positive for M. bovis, while the results for the LCx assay and the histological method were positive in 48 (45, 3) and 24 (20, 4) samples, respectively. None of the samples from cattle from certified tuberculosis-free herds were positive by any of the procedures. The results obtained with the LCx assay, compared with the culture procedure, regarded as the gold standard among the diagnostic techniques, gave a specificity of 91.6% and sensitivity of 90.5%. Although the sensitivity of LCx was suboptimal, DNA of M. bovis was detected in 81.8% of the skin test-positive animals. Amplification techniques could provide a rapid and reasonably reliable tool for detecting bovine tuberculosis.

Animals↗
PubMed2004

Optimization of 13C direct detection NMR methods.

(13)C-detected experiments are still limited by their inherently lower sensitivity, as compared to the equivalent (1)H-detected experiments. Improving the sensitivity of (13)C detection methods remains a significant area of NMR research that may provide better means for studying large macromolecular systems by NMR. In this communication, we show that (13)C-detected experiments are less sensitive to the salt concentration of the sample solution than (1)H-detected experiments. In addition, acquisition can be started with anti-phase coherence, resulting in higher sensitivity due to the elimination of the final INEPT transfer step.

Carbon Isotopes↗