Research of the Properties of Gadolinium Stearate by EPR Spectroscopy

Russian Journal of Coordination Chemistry - Gadolinium stearate is synthesized in the form of a micropowder. Its composition is determined by elemental and energy dispersive X-ray analyses:...     

Determination of the oligomeric number and intermolecular distances of membrane protein assemblies by anisotropic 1H-driven spin diffusion NMR spectroscopy

Determination of the high-resolution quaternary structure of oligomeric membrane proteins requires knowledge of both the oligomeric number and intermolecular distances. The centerband-only detection of exchange (CODEX) technique has been shown to enable the extraction of the oligomeric number through the equilibrium exchange intensity at long mixing times. To obtain quantitative distances, we now provide an analysis of the mixing-time-dependent CODEX intensities using the 1H-driven spin diffusion theory. The exchange curve is fit to a rate equation, where the rate constants are proportional to the square of the dipolar coupling and the spectral overlap integral between the exchanging spins. Using a number of 13C- and 19F-labeled crystalline model compounds with known intermolecular distances, we empirically determined the overlap integrals of 13C and 19F CODEX for specific spinning speeds and chemical shift anisotropies. These consensus overlap integral values can be applied to structurally unknown systems to determine distances. Applying the 19F CODEX experiment and analysis, we studied the transmembrane peptide of the M2 protein (M2TMP) of influenza A virus bound to 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers. The experiment proved for the first time that M2TMP associates as tetramers in lipid bilayers, similar to its oligomeric state in detergent micelles. Moreover, the nearest-neighbor interhelical F-F distance between (4-19F)Phe30 is 7.9-9.5 angstroms. This distance constrains the orientation and the packing of the helices in the tetrameric bundle and supports the structural model derived from previous solid-state NMR 15N orientational data. Thus, the CODEX technique presents a general method for determining the oligomeric number and intermolecular distances in the approximately 10 angstroms range in membrane proteins and other complex biological assemblies.     

Biomimetic dual templating of silica by polysaccharide/protein assemblies

The control of silica growth by living organisms such as diatoms is known to involve the templating effect of several biomolecules working concomitantly. However, until now, biomimetic studies involving model molecules have mainly been performed with single templates. We show here that the addition of two biopolymers, gelatin and alginic acid, to silicate solutions allows the formation of complex structures resulting from the combined templating effect of both components at different scales. Gelatin is able to activate silica formation resulting in hybrid aggregates at the nanoscale. Alginic acid does not interfere with silica condensation but is able to control silica morphology through the assembly of these gelatin-silica aggregates at the microscale. For all materials, calcination up to 700 degrees C degrades the polymer component of the hybrid material and opens macroporosity in the silica network. In parallel, the high thermal stability of gelatin allows a good preservation of initial silica nanoparticle size upon heating whereas a coarsening process is observed in the sole presence of alginate. These results correlate well with previous models of biosilicification and suggest that the use of multiple templates is a suitable approach to elaborate more complex silica architectures.     

Selection rules and symmetry relations for four-wave mixing measurements of uniaxial assemblies

Uniaxial systems represent the next lowest symmetry below isotropic and are ubiquitous. The objective of the present work is to present a systematic foundation for interpreting polarization-dependent four-wave mixing measurements of oriented and aligned assemblies. Orientational averages connecting the molecular frame to the macroscopic frame in uniaxial assemblies were derived for several common molecular symmetry groups for coherent anti-Stokes Raman spectroscopy (CARS) measurements, coherent anti-Stokes two-photon spectroscopy (CATS) probing electronic transitions, resonant two-photon absorption (2PA), and traditional Raman measurements. First, the complete set of orientational averages connecting the molecular and macroscopic frames was compiled for the most general case of C1 molecular symmetry. Then, the orientational averages of a select few commonly occurring molecular symmetry groups (Cs, C2, C2v, and C3v) were explored in greater detail to illustrate the approach and to facilitate the interpretation of routine experimental measurements. One outcome of this analysis is the prediction of efficient electric dipole-allowed chiral-specific four-wave mixing in uniaxially oriented media.     

Bioorthogonal chemical tagging of protein cholesterylation in living cells

We report the first chemical probe for bioorthogonal chemical tagging of post-translationally cholesterylated proteins with an azide in living cells. This enables rapid multiplexed fluorescence detection and affinity labelling of protein cholesterylation, as exemplified by Sonic hedgehog protein, opening up new approaches for the de novo identification of cholesterylated proteins.     

An EPR method for probing surface magnetic fields, dipolar distances, and magnetization fluctuations in single molecule magnets

We outline a spectroscopic method for probing the effective magnetic field B on the surface of crystals of the single molecule magnet (SMM) [(C6H15N3)6Fe8(mu3-O)2(mu2-OH)12]Br7(H2O)Br.H2O, (Fe8Br8). This technique utilizes the line shape changes in the EPR spectra of the organic radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) (g = 2.0036, single peak) adsorbed onto the sample. The temperature dependence of the EPR line shifts scale with the sample's magnetization as measured by a SQUID magnetometer. Analysis of the line shape in terms of dipolar coupling between the DPPH and the SMM molecules on the surface, yields their average dipolar distance. The method's potential for measuring the magnetization fluctuation dynamics is briefly pointed out using the SMM [Mn12O12(CH3COO)16(H2O)4].2CH3COOH.4H2O (Mn12-acetate).     

Energy transfer dynamics in light-harvesting assemblies templated by the tobacco mosaic virus coat protein

Picosecond time-resolved fluorescence spectroscopy was used to characterize energy transfer between chromophores displayed on a rod assembly of tobacco mosaic virus coat protein. The incorporation of donor chromophores with broad and overlapping absorption and emission spectra creates an "antenna" with a large absorption cross section, which can convey excitation energy over large distances before transfer to an acceptor chromophore. The possibility for both donor-to-donor and donor-to-acceptor transfer results in complex kinetic behavior at any single wavelength. Thus, to describe the various pathways of energy transfer within this system accurately, a global lifetime analysis was performed to obtain decay associated spectra. We found the energy transfer from donor to acceptor chromophores occurs in 187 ps with an efficiency of 36%. A faster decay component of 70 ps was also observed from global lifetime analysis and is attributed to donor-to-donor transfer. Although more efficient three-chromophore systems have been demonstrated, a two-chromophore system was studied here to facilitate analysis.     

A minimal cavity and its capacitive coupling for in vivo EPR measurements on mice.

A very simple ring resonator for in vivo L-band EPR spectroscopy was built and characterised. It employs a special capacitive coupling that allows measurents to be made on large biological samples which are not possible with other resonators. In spite of its intrinsic low Q it has a sensitivity almost equivalent to that obtained from high Q resonators. These features were tested down to a nitroxide concentration of 10 μM in high conductivity phantoms.     

Magic angle spinning NMR experiments for structural studies of differentially enriched protein interfaces and protein assemblies

Protein-protein interactions play vital roles in numerous biological processes. These interactions often result in formation of insoluble and noncrystalline protein assemblies. Solid-state NMR spectroscopy is rapidly emerging as a premier method for structural analysis of such systems. We introduce a family of two-dimensional magic angle spinning (MAS) NMR experiments for structural studies of differentially isotopically enriched protein assemblies. Using 1-73((13)C,(15)N)/74-108((15)N) labeled thioredoxin reassembly, we demonstrate that dipolar dephasing followed by proton-assisted heteronuclear magnetization transfer yields long-range (15)N-(13)C correlations arising exclusively from the interfaces formed by the pair of differentially enriched complementary fragments of thioredoxin. Incorporation of dipolar dephasing into the (15)N proton-driven spin diffusion and into the (1)H-(15)N FSLG-HETCOR sequences permits (1)H and (15)N resonance assignments of the 74-108((15)N) enriched C-terminal fragment of thioredoxin alone. The differential isotopic labeling scheme and the NMR experiments demonstrated here allow for structural analysis of both the interface and each interacting protein. Isotope editing of the magnetization transfers results in spectral simplification, and therefore larger protein assemblies are expected to be amenable to these experiments.     

Preparation and evaluation of the quality of standards for quantitative EPR measurements of spin concentration

Summary In continuation of previous work on the preparation and evaluation of standards for EPR measurements, investigations are described of vanadyl standards diluted with K₂SO₄. This included the preparation of five standards, checking their quality by statistical methods and determining the vanadium content by differential pulse polarography.     

Natural intramolecular isotope measurements in physiology: elements of the case for an effort toward high-precision position-specific isotope analysis

Chemical information available in organisms can be categorized into three major domains, macromolecular, small molecules, and isotope ratios. Information about physiological state is commonly obtained by qualitative and quantitative analysis in the macromolecular and small molecule domains. Genomics and proteomics are emerging approaches to analysis of macromolecules, and both areas yield definitive information on present physiological state. There is relatively little record of past physiological states of the individual available in these domains. Natural isotopic variability, particularly on an intramolecular level, is likely to retain more physiological history. Because of ubiquitous isotopic fractionation, every stereochemically unique position in every molecule has an isotope ratio that reflects the processes of synthesis and degradation. This fact highlights a vast amount of organismal chemical information that is essentially unstudied. Isotope measurements can be classified according to the chemical complexity of the analyte into bulk, compound-specific, and position-specific or intramolecular levels. Recent advances in analysis of isotope ratios are transforming natural science, and particularly answering questions about ecosystems using bulk methods; however, they have had relatively little impact on physiology. This may be because the vast complexities of physiological questions demand very selective information available in position-specific isotope analysis (PSIA). The relatively few high-precision PSIA studies, based on isotope ratio mass spectrometry (IRMS), have revealed intramolecular isotope ratio differences in pivotal physiological compounds including amino acids, glucose, glycerol, acetate, fatty acids, and purines. The majority of these analyses have been accomplished by laborious offline methods; however, recent advances in instrumentation presage rapid PSIA that will be necessary to attack real physiological problems. Gas-phase pyrolysis has been shown to be an effective method to determine (13)C/(12)C at high precision for molecular fragments, and technologies to extend C-based PSIA to N and other organic elements are emerging. Two related efforts are warranted, (a) development of rapid, convenient, and sensitive methods for high-precision PSIA, a necessary precursor to (b) a concerted investigation into the relationship of metabolic state to intramolecular isotope ratio. Inherent in this latter goal is the need to identify long-lived molecules in long-lived cells that retain a record of early isotopic conditions, as has been shown for post-mortem human neuronal DNA. Using known metabolic precursor-product relationships between intramolecular positions, future studies of physiological isotope fractionation should reveal the relationship of diet and environment to observed isotope ratio. This science of isotope physiology, or simply isotopics, should add an important tool for elucidation of early factors that effect later health, probably the most difficult class of biomedical issues.     

Characterization of the B6 phase by dielectric measurements

Dielectric measurements on a sample from banana-shaped molecules were carried out between 1Hz and 10MHz. The sample exhibits at higher temperatures the B₆ phase and at lower temperatures the B₁ phase. No difference in the dynamical behaviour between these two phases with respect to the relaxation times, dielectric increments and distribution parameters could be detected. Both phases show a negative dipole correlation for the reorientation of the dipoles in the stiff middle part about the long axes.     

Plasmon coupling of R6G-linked gold nanoparticle assemblies for surface-enhanced Raman spectroscopy

This paper presents the study of surface-enhanced Raman scattering (SERS) for Rhodamine 6G (R6G)-linked gold (Au) assemblies. The nano-assemblies fabricated with Au nanoparticles, having diameters (D) of ～5–40 nm, possessed interparticle gaps of ～0.03–0.2D, respectively. The R6G-linked Au assemblies displayed weak spectral shifts of localized surface plasmon resonance (LSPR) mode at shorter wavelength region and large fractional red-shifts Δλ ～ 0.07–0.47λ_o at longer wavelength region. The resonant spectral coupling of the nano-assemblies LSPR mode at the longer wavelength region and the optical excitation at ～785 nm, in addition to the highly localized hot spots, could support the large SERS enhancement of R6G-linked Au assemblies.     

Characterization of the B6 phase by dielectric measurements

Dielectric measurements on a sample from banana-shaped molecules were carried out between 1Hz and 10MHz. The sample exhibits at higher temperatures the B₆ phase and at lower temperatures the B₁ phase. No difference in the dynamical behaviour between these two phases with respect to the relaxation times, dielectric increments and distribution parameters could be detected. Both phases show a negative dipole correlation for the reorientation of the dipoles in the stiff middle part about the long axes.     

EPR distance measurements support a model for long-range radical initiation in E. coli ribonucleotide reductase

The class I E. coli ribonucleotide reductase, composed of homodimers of R1 and R2, catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. The reduction process involves the tyrosyl radical on R2 that generates a transient thiyl radical on R1 over a proposed distance of 35 A. A mechanism-based inhibitor, 2'-azido-2'-deoxyuridine-5'-diphosphate, that reduces the tyrosyl radical on R2 and forms a nitrogen-centered radical on R1 has provided a method to measure the diagonal distance between the two subunits. PELDOR and DQC paramagnetic resonance methods give rise to a distance of 48 A, similar to that calculated from a docking model of the R1 and R2 structures.     

Nested Arg-specific bifunctional crosslinkers for MS-based structural analysis of proteins and protein assemblies

The combination of chemical probing and high-resolution mass spectrometry constitutes a powerful alternative for the structural elucidation of biomolecules possessing unfavorable size, solubility, and flexibility. We have developed nested Arg-specific bifunctional crosslinkers to obtain complementary information to typical Cys- and Lys-specific reagents available on the market. The structures of 1,4-phenyl-diglyoxal (PDG) and 4,4′-biphenyl-diglyoxal (BDG) include two identical 1,2-dicarbonyl functions capable of reacting with the guanido group of Arg residues in proteins, as well as the base-pairing face of guanine in nucleic acids. The reactive functions are separated by modular spacers consisting of one or two benzene rings, which confer greater rigidity to the crosslinker structure than it is afforded by typical aliphatic spacers. Analysis by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has shown that the probes provide both mono- and bifunctional products with model protein substrates, which are stabilized by the formation of diester derivatives in the presence of borate buffer. The identification of crosslinked sites was accomplished by employing complementary proteolytic procedures and peptide mapping by ESI-FTICR. The results showed excellent correlation with the solvent accessibility and structural context of susceptible residues, and highlighted the significance of possible dynamic effects in determining the outcome of crosslinking reactions. The application of nested reagents with different spacing has provided a new tool for experimentally recognizing flexible regions that may be involved in prominent dynamics in solution. The development of new bifunctional crosslinkers with diverse target specificity and different bridging spans is expected to facilitate the structure elucidation of progressively larger biomolecular assemblies by increasing the number and diversity of spatial constraints available for triangulating the position of crosslinked structures in the three dimensions.