Peptidic "Molecular Beacon" for Collagen.

Collagen-mimetic peptides (CMP) have been invaluable tools for understanding the structure and function of collagen, which is the most abundant protein in animals. CMPs have also been developed as probes that detect damaged collagen because of the specificity required to form a collagen triple helix. These probes are not, however, ratiometric. Here, we used EPR spectroscopy to determine the end-to-end distances of CMPs that do not form stable homotrimeric helices. We found that those distances are shorter than the distances in the context of a collagen triple helix, suggesting their potential utility as a "molecular beacon" and guiding the choice and location of a pendant fluorophore-quencher pair. We then showed that a molecular beacon based on a glycine-(2S,4S)-4-fluoroproline-(2S,4R)-4-hydroxyproline tripeptide repeat and EDANS-DABCYL pair enabled the ratiometric detection of its binding to both other CMPs and natural mammalian collagen. These results provide guidance for the development of a new modality for detecting damaged collagen in physiological settings.

Frequency-Swept Integrated Solid Effect.

The efficiency of continuous wave dynamic nuclear polarization (DNP) experiments decreases at the high magnetic fields used in contemporary high-resolution NMR applications. To recover the expected signal enhancements from DNP, we explored time domain experiments such as NOVEL which matches the electron Rabi frequency to the nuclear Larmor frequency to mediate polarization transfer. However, satisfying this matching condition at high frequencies is technically demanding. As an alternative we report here frequency-swept integrated solid effect (FS-ISE) experiments that allow low power sweeps of the exciting microwave frequencies to constructively integrate the negative and positive polarizations of the solid effect, thereby producing a polarization efficiency comparable to (±10 % difference) NOVEL. Finally, the microwave frequency modulation results in field profiles that exhibit new features that we coin the "stretched" solid effect.

Tailoring of Polarizing Agents in the bTurea Series for Cross-Effect Dynamic Nuclear Polarization in Aqueous Media.

A series of 18 nitroxide biradicals derived from bTurea has been prepared, and their enhancement factors ɛ ((1)H) in cross-effect dynamic nuclear polarization (CE DNP) NMR experiments at 9.4 and 14.1 T and 100 K in a DNP-optimized glycerol/water matrix ("DNP juice") have been studied. We observe that ɛ ((1)H) is strongly correlated with the substituents on the polarizing agents, and its trend is discussed in terms of different molecular parameters: solubility, average e-e distance, relative orientation of the nitroxide moieties, and electron spin relaxation times. We show that too short an e-e distance or too long a T1e can dramatically limit ɛ ((1)H). Our study also shows that the molecular structure of AMUPol is not optimal and its ɛ ((1)H) could be further improved through stronger interaction with the glassy matrix and a better orientation of the TEMPO moieties. A new AMUPol derivative introduced here provides a better ɛ ((1)H) than AMUPol itself (by a factor of ca. 1.2).

Probing the Noninnocent π-Bonding Influence of N-Carboxyamidoquinolate Ligands on the Light Harvesting and Redox Properties of Ruthenium Polypyridyl Complexes.

Electronic and photophysical characterization is presented for a series of bis-heteroleptic [Ru(bpy)2(R-CAQN)](+) complexes where CAQN is a bidentate N-(carboxyaryl)amidoquinolate ligand and the aryl substituent R = p-tolyl, p-fluorobenzene, p-trifluoromethylbenzene, 3,5-bis(trifluoromethyl)benzene, or 4-methoxy-2,3,5,6-tetrafluorobenzene. Characterized by a strong noninnocent Ru(dπ)-CAQN(π) bonding interaction, density functional theory (DFT) analysis is used to estimate the contribution of both atomic Ru(dπ) and ligand CAQN(π) manifolds to the frontier molecular orbitals of these complexes. UV-vis absorption and emission studies are presented where the noninnocent Ru(dπ)-CAQN(π) bonding scheme plays a major role in defining complex electronic and photophysical properties. Oxidation potentials are tuned over a range of 0.92 V with respect to the [Ru(bpy)3](2+) reference system, hereafter referred to as 1(2+), by varying the degree of R-CAQN fluorination while maintaining consistently strong and panchromatic visible absorption properties. Electron paramagnetic resonance (EPR) spectroscopy is employed to experimentally map delocalization of the unpaired electron/electron-hole within the delocalized Ru(dπ)-CAQN(π) singly occupied valence molecular orbital of the one-electron oxidized complexes. EPR data is complemented experimentally by UV-vis-NIR spectroelectrochemistry, and computationally by molecular orbital Mulliken contributions and spin-density analysis. It is ultimately demonstrated that the CAQN ligand framework provides a simple yet broad synthetic platform in the design of redox-active transition metal chromophores with a range of electronic and spectroscopic characteristics hinting at the diversity and potential of these complexes toward photochemical and catalytic applications.

A novel copper(II) complex identified as a potent drug against colorectal and breast cancer cells and as a poison inhibitor for human topoisomerase IIα.

A novel complex, [Cu(acetylethTSC)Cl]Cl•0.25C2H5OH 1 (where acetylethTSC = (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide), was shown to have anti-proliferative activity against various colon and aggressive breast cancer cell lines. In vitro studies showed that complex 1 acted as a poison inhibitor of human topoisomerase IIα, which may account for the observed anti-cancer effects.

EPR detection of two protein-associated ubiquinone components (SQ(Nf) and SQ(Ns)) in the membrane in situ and in proteoliposomes of isolated bovine heart complex I.

The success of Sazanov's group in determining the X-ray structure of the whole bacterial complex I is a great contribution to the progress of complex I research. In this mini-review of 35years' history of my laboratory and collaborators, we characterized the function of protein-associated semiquinone molecules in the proton-pumping mechanism in complex I (NADH-quinone oxidoreductase). We have constructed most of the frame work of our hypothesis, utilizing EPR techniques before the X-ray structures of complex I were reported by Sazanov's and Brandt's groups. One of the semiquinones (SQ(Nf)) is extremely sensitive to a proton motive force imposed on the energy-transducing membrane, while the other (SQ(Ns)) is insensitive. Their sensitivity to rotenone inhibition also differs. These differences were exploited using tightly coupled bovine heart submitochondrial particles with a high respiratory control ratio (>8). We determined the distance between SQ(Nf) and iron-sulfur cluster N2 on the basis of their direct spin-spin interaction. We are extending this line of work using reconstituted bovine heart complex I proteoliposomes which shows a respiratory control ratio >5. Two frontier research groups support our view point based on their mutagenesis studies. High frequency (33.9GHz; Q-band) EPR experiments appear to favor our two-semiquinone model. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Nonaqueous synthesis of TiO2 nanocrystals using TiF4 to engineer morphology, oxygen vacancy concentration, and photocatalytic activity.

Control over faceting in nanocrystals (NCs) is pivotal for many applications, but most notably when investigating catalytic reactions which occur on the surfaces of nanostructures. Anatase titanium dioxide (TiO(2)) is one of the most studied photocatalysts, but the shape dependence of its activity has not yet been satisfactorily investigated and many questions still remain unanswered. We report the nonaqueous surfactant-assisted synthesis of highly uniform anatase TiO(2) NCs with tailorable morphology in the 10-100 nm size regime, prepared through a seeded growth technique. Introduction of titanium(IV) fluoride (TiF(4)) preferentially exposes the {001} facet of anatase through in situ release of hydrofluoric acid (HF), allowing for the formation of uniform anatase NCs based on the truncated tetragonal bipyramidal geometry. A method is described to engineer the percentage of {001} and {101} facets through the choice of cosurfactant and titanium precursor. X-ray diffraction studies are performed in conjunction with simulation to determine an average NC dimension which correlates with results obtained using electron microscopy. In addition to altering the particle shape, the introduction of TiF(4) into the synthesis results in TiO(2) NCs that are blue in color and display a broad visible/NIR absorbance which peaks in the infrared (λ(max) ≈ 3400 nm). The blue color results from oxygen vacancies formed in the presence of fluorine, as indicated by electron paramagnetic resonance (EPR) and X-ray photoelectron spectroscopy (XPS) studies. The surfactants on the surface of the NCs are removed through a simple ligand exchange procedure, allowing the shape dependence of photocatalytic hydrogen evolution to be studied using monodisperse TiO(2) NCs. Preliminary experiments on the photoreforming of methanol, employed as a model sacrificial agent, on platinized samples resulted in high volumes of evolved hydrogen (up to 2.1 mmol h(-1) g(-1)) under simulated solar illumination. Remarkably, the data suggest that, under our experimental conditions, the {101} facets of anatase are more active than the {001}.

A slowly relaxing rigid biradical for efficient dynamic nuclear polarization surface-enhanced NMR spectroscopy: expeditious characterization of functional group manipulation in hybrid materials.

A new nitroxide-based biradical having a long electron spin-lattice relaxation time (T(1e)) has been developed as an exogenous polarization source for DNP solid-state NMR experiments. The performance of this new biradical is demonstrated on hybrid silica-based mesostructured materials impregnated with 1,1,2,2-tetrachloroethane radical containing solutions, as well as in frozen bulk solutions, yielding DNP enhancement factors (ε) of over 100 at a magnetic field of 9.4 T and sample temperatures of ~100 K. The effects of radical concentration on the DNP enhancement factors and on the overall sensitivity enhancements (Σ(†)) are reported. The relatively high DNP efficiency of the biradical is attributed to an increased T(1e), which enables more effective saturation of the electron resonance. This new biradical is shown to outperform the polarizing agents used so far in DNP surface-enhanced NMR spectroscopy of materials, yielding a 113-fold increase in overall sensitivity for silicon-29 CPMAS spectra as compared to conventional NMR experiments at room temperature. This results in a reduction in experimental times by a factor >12,700, making the acquisition of (13)C and (15)N one- and two-dimensional NMR spectra at natural isotopic abundance rapid (hours). It has been used here to monitor a series of chemical reactions carried out on the surface functionalities of a hybrid organic-silica material.

Solid-state dynamic nuclear polarization at 263 GHz: spectrometer design and experimental results.

Dynamic Nuclear Polarization (DNP) experiments transfer polarization from electron spins to nuclear spins with microwave irradiation of the electron spins for enhanced sensitivity in nuclear magnetic resonance (NMR) spectroscopy. Design and testing of a spectrometer for magic angle spinning (MAS) DNP experiments at 263 GHz microwave frequency, 400 MHz (1)H frequency is described. Microwaves are generated by a novel continuous-wave gyrotron, transmitted to the NMR probe via a transmission line, and irradiated on a 3.2 mm rotor for MAS DNP experiments. DNP signal enhancements of up to 80 have been measured at 95 K on urea and proline in water-glycerol with the biradical polarizing agent TOTAPOL. We characterize the experimental parameters affecting the DNP efficiency: the magnetic field dependence, temperature dependence and polarization build-up times, microwave power dependence, sample heating effects, and spinning frequency dependence of the DNP signal enhancement. Stable system operation, including DNP performance, is also demonstrated over a 36 h period.

Adiabatic Solid Effect.

The solid effect (SE) is a two spin dynamic nuclear polarization (DNP) mechanism that enhances the sensitivity in NMR experiments by irradiation of the electron-nuclear spin transitions with continuous wave (CW) microwaves at ω0S ± ω0I, where ω0S and ω0I are electron and nuclear Larmor frequencies, respectively. Using trityl (OX063), dispersed in a 60/40 glycerol/water mixture at 80 K, as a polarizing agent, we show here that application of a chirped microwave pulse, with a bandwidth comparable to the EPR line width applied at the SE matching condition, improves the enhancement by a factor of 2.4 over the CW method. Furthermore, the chirped pulse yields an enhancement that is ∼20% larger than obtained with the ramped-amplitude NOVEL (RA-NOVEL), which to date has achieved the largest enhancements in time domain DNP experiments. Numerical simulations suggest that the spins follow an adiabatic trajectory during the polarization transfer; hence, we denote this sequence as an adiabatic solid effect (ASE). We foresee that ASE will be a practical pulsed DNP experiment to be implemented at higher static magnetic fields due to the moderate power requirement. In particular, the ASE uses only 13% of the maximum microwave power required for RA-NOVEL.

Time-optimized pulsed dynamic nuclear polarization.

Pulsed dynamic nuclear polarization (DNP) techniques can accomplish electron-nuclear polarization transfer efficiently with an enhancement factor that is independent of the Zeeman field. However, they often require large Rabi frequencies and, therefore, high-power microwave irradiation. Here, we propose a new low-power DNP sequence for static samples that is composed of a train of microwave pulses of length τp spaced with delays d. A particularly robust DNP condition using a period τm = τp + d set to ~1.25 times the Larmor period τLarmor is investigated which is a time-optimized pulsed DNP sequence (TOP-DNP). At 0.35 T, we obtained an enhancement of ~200 using TOP-DNP compared to ~172 with nuclear spin orientation via electron spin locking (NOVEL), a commonly used pulsed DNP sequence, while using only ~7% microwave power required for NOVEL. Experimental data and simulations at higher fields suggest a field-independent enhancement factor, as predicted by the effective Hamiltonian.

Structure and dynamics of the force-generating domain of myosin probed by multifrequency electron paramagnetic resonance.

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.

On the Use of Q-Band Double Electron-Electron Resonance To Resolve the Relative Orientations of Two Double Histidine-Bound Cu2+ Ions in a Protein.

In this work, we explore the potential of a rigid Cu2+ spin-labeling technique, the double histidine (dHis) motif, along with Q-band electron paramagnetic resonance to report on the relative orientations of the spin labels. We show that the precision of the dHis motif, coupled with the sensitivity and resolution of Q-band frequencies, may allow for the straightforward determination of the relative orientation of the dHis-Cu2+ labels using double electron-electron resonance (DEER). We performed Q-band DEER measurements at different magnetic fields on a protein containing two dHis Cu2+ sites. These measurements exhibited orientational selectivity such that each discrete magnetic field yielded a unique DEER signal. We determined the relative orientation of the two metal centers by simulating the orientationally selective DEER data. These relative orientations were validated by visual analysis of the protein crystal structure modified with dHis sites. The simple visual analysis was shown to agree well with the angular values determined via simulation of the experimental data. The combination of the dHis-Cu2+ motif along with the advantages of the Q-band can aid in the accurate measurement of protein structural and conformational dynamics.

Multifrequency EPR studies on the Mn(II) centers of oxalate decarboxylase.

Oxalate decarboxylase from Bacillus subtilis is composed of two cupin domains, each of which contains a Mn(II) ion coordinated by four identical conserved residues. The similarity between the two Mn(II) sites has precluded previous attempts to distinguish them spectroscopically and complicated efforts to understand the catalytic mechanism. A multifrequency cw-EPR approach has now enabled us to show that the two Mn ions can be distinguished on the basis of their differing fine structure parameters and to observe that acetate and formate bind to Mn(II) in only one of the two sites. The EPR evidence is consistent with the hypothesis that this Mn-binding site is located in the N-terminal domain, in agreement with predictions based on a recent X-ray structure of the enzyme.

3,5-Di-tert-butyl-3'-(N-tert-butyl-N-aminoxy)-4-oxybiphenyl: A Heterospin Diradical with Temperature Dependent Behavior.

The title diradical was synthesized and investigated by ESR and UV-vis spectroscopy. It was found to have a lifetime of weeks even in the presence of oxygen, and even survives brief heating in toluene up to about 60 °C. In the UV-vis spectrum, the diradical showed reversible thermochromic behavior in the -10 to 50 °C range. In the ESR spectrum, hyperfine analysis showed nearly isolated behavior by the phenoxyl and nitroxide spin carrying units. Upon warming, additional, broad lines appeared at the expense of the lower temperature lines. This temperature behavior was reversible over the 0 to 50 °C range, so long as the raised temperatures were not maintained for long time periods. The spectral behavior is interpreted as being due to temperature-dependent conformational effects on the exchange coupling between the spin carrying units, i.e., J-modulated exchange behavior.

Skew projection of echo-detected EPR spectra for increased sensitivity and resolution.

The measurement of EPR spectra during pulsed EPR experiments is commonly accomplished by recording the integral of the electron spin echo as the applied magnetic field is stepped through the spectrum. This approach to echo-detected EPR spectral measurement (ED-EPR) limits sensitivity and spectral resolution and can cause gross distortions in the resulting spectra because some of the information present in the electron spin echo is discarded in such measurements. However, Fourier transformation of echo shapes measured at a series of magnetic field values followed by skew projection onto either a magnetic field or resonance frequency axis can increase both spectral resolution and sensitivity without the need to trade one against the other. Examples of skew-projected spectra with single crystals, glasses and powders show resolution improvements as large as a factor of seven with sensitivity increases of as much as a factor of five.

Preliminary anti-cancer photodynamic therapeutic in vitro studies with mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes.

We report the synthesis and characterisation of mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes, which were used as potential photodynamic therapeutic agents for melanoma cell growth inhibition. The novel complexes, [Ru(pbt)2(phen2DTT)](PF6)2·1.5H2O 1 (where phen2DTT = 1,4-bis(1,10-phenanthrolin-5-ylsulfanyl)butane-2,3-diol and pbt = 2-(2'-pyridyl)benzothiazole) and [Ru(pbt)2(tpphz)](PF6)2·3H2O 2 (where tpphz = tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine) were synthesised and characterised. Compound 1 was reacted with [VO(sal-L-tryp)(H2O)] (where sal-L-tryp = N-salicylidene-L-tryptophanate) to produce [Ru(pbt)2(phen2DTT)VO(sal-L-tryp)](PF6)2·5H2O 4; while [VO(sal-L-tryp)(H2O)] was reacted with compound 2 to produce [Ru(pbt)2(tpphz)VO(sal-L-tryp)](PF6)2·6H2O 3. All complexes were characterised by elemental analysis, HRMS, ESI MS, UV-visible absorption, ESR spectroscopy, and cyclic voltammetry, where appropriate. In vitro cell toxicity studies (with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay) via dark and light reaction conditions were carried out with sodium diaqua-4,4',4'',4''' tetrasulfophthalocyaninecobaltate(II) (Na4[Co(tspc)(H2O)2]), [VO(sal-L-tryp)(phen)]·H2O, and the chloride salts of complexes 3 and 4. Such studies involved A431, human epidermoid carcinoma cells; human amelanotic malignant melanoma cells; and HFF, non-cancerous human skin fibroblast cells. Both chloride salts of complexes 3 and 4 were found to be more toxic to melanoma cells than to non-cancerous fibroblast cells, and preferentially led to apoptosis of the melanoma cells over non-cancerous skin cells. The anti-cancer property of the chloride salts of complexes 3 and 4 was further enhanced when treated cells were exposed to light, while no such effect was observed on non-cancerous skin fibroblast cells. ESR and (51)V NMR spectroscopic studies were also used to assess the stability of the chloride salts of complexes 3 and 4 in aqueous media at pH 7.19. This research illustrates the potential for using mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes to fight skin cancer.

Enhancement of the electron spin resonance of single-walled carbon nanotubes by oxygen removal.

We have observed a nearly 4-fold increase in the electron spin resonance (ESR) signal from an ensemble of single-walled carbon nanotubes (SWCNTs) due to oxygen desorption. By performing temperature-dependent ESR spectroscopy both before and after thermal annealing, we found that the ESR in SWCNTs can be reversibly altered via the molecular oxygen content in the samples. Independent of the presence of adsorbed oxygen, a Curie law (spin susceptibility ∝ 1/T) is seen from ~4 to 300 K, indicating that the probed spins are finite-level species. For both the pre-annealed and post-annealed sample conditions, the ESR line width decreased as the temperature was increased, a phenomenon we identify as motional narrowing. From the temperature dependence of the line width, we extracted an estimate of the intertube hopping energy; for both sample conditions, we found this hopping energy to be ~1.2 meV. Since the spin hopping energy changes only slightly when oxygen is desorbed, we conclude that only the spin susceptibility, not spin transport, is affected by the presence of physisorbed molecular oxygen in SWCNT ensembles. Surprisingly, no line width change is observed when the amount of oxygen in the SWCNT sample is altered, contrary to other carbonaceous systems and certain 1D conducting polymers. We hypothesize that physisorbed molecular oxygen acts as an acceptor (p-type), compensating the donor-like (n-type) defects that are responsible for the ESR signal in bulk SWCNTs.