Matrix-assisted diffusion-ordered spectroscopy:mixture resolution by NMR using SDSmicelles

Diffusion-ordered spectroscopy (DOSY) is a powerful technique for mixture analysis, but in its basic form it cannot separate the component spectra for species with very similar diffusion coefficients. It has been recently demonstrated that the component spectra of a mixture of isomers with nearly identical diffusion coefficients (the three dihydroxybenzenes) can be resolved using matrix-assisted DOSY (MAD), in which diffusion is perturbed by the addition of a co-solute such as a surfactant [R. Evans, S. Haiber, M. Nilsson, G. A. Morris, Anal. Chem. 2009, 81, 4548-4550]. However, little is known about the conditions required for such a separation, for example, the concentrations and concentration ratios of surfactant and solutes. The aim of this study was to explore the concentration range over whichmatrix-assisted DOSY using the surfactant SDS can achieve diffusion resolution of a simple model set of isomers, the monomethoxyphenols. The results show that the separation is remarkably robust with respect to both the concentrations and the concentration ratios of surfactant and solutes, supporting the idea that MAD may become a valuable tool formixture analysis. © 2010 John Wiley & Sons, Ltd.

Proton nuclear magnetic resonance investigation of the native and modified active site structure of heme proteins

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. ^ During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. ^ Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. ^ The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN - complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.^

Optimization of wireless power transfer via magnetic resonance in different media

A wide range of non-destructive testing (NDT) methods for the monitoring the health of concrete structure has been studied for several years. The recent rapid evolution of wireless sensor network (WSN) technologies has resulted in the development of sensing elements that can be embedded in concrete, to monitor the health of infrastructure, collect and report valuable related data. The monitoring system can potentially decrease the high installation time and reduce maintenance cost associated with wired monitoring systems. The monitoring sensors need to operate for a long period of time, but sensors batteries have a finite life span. Hence, novel wireless powering methods must be devised. The optimization of wireless power transfer via Strongly Coupled Magnetic Resonance (SCMR) to sensors embedded in concrete is studied here. First, we analytically derive the optimal geometric parameters for transmission of power in the air. This specifically leads to the identification of the local and global optimization parameters and conditions, it was validated through electromagnetic simulations. Second, the optimum conditions were employed in the model for propagation of energy through plain and reinforced concrete at different humidity conditions, and frequencies with extended Debye's model. This analysis leads to the conclusion that SCMR can be used to efficiently power sensors in plain and reinforced concrete at different humidity levels and depth, also validated through electromagnetic simulations. The optimization of wireless power transmission via SMCR to Wearable and Implantable Medical Device (WIMD) are also explored. The optimum conditions from the analytics were used in the model for propagation of energy through different human tissues. This analysis shows that SCMR can be used to efficiently transfer power to sensors in human tissue without overheating through electromagnetic simulations, as excessive power might result in overheating of the tissue. Standard SCMR is sensitive to misalignment; both 2-loops and 3-loops SCMR with misalignment-insensitive performances are presented. The power transfer efficiencies above 50% was achieved over the complete misalignment range of 0°-90° and dramatically better than typical SCMR with efficiencies less than 10% in extreme misalignment topologies.

Nuclear magnetic resonance spectroscopic and computational investigations of chloroperoxidase catalyzed regio- and enantio-selective transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. ^ To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. ^ To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O 2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.^

Determination of Biomolecular Interdomain Motions using Nuclear Magnetic Resonance

Biological macromolecules can rearrange interdomain orientations when binding to various partners. Interdomain dynamics serve as a molecular mechanism to guide the transitions between orientations. However, our understanding of interdomain dynamics is limited because a useful description of interdomain motions requires an estimate of the probabilities of interdomain conformations, increasing complexity of the problem.

Staphylococcal protein A (SpA) has five tandem protein-binding domains and four interdomain linkers. The domains enable Staphylococcus aureus to evade the host immune system by binding to multiple host proteins including antibodies. Here, I present a study of the interdomain motions of two adjacent domains in SpA. NMR spin relaxation experiments identified a 6-residue flexible interdomain linker and interdomain motions. To quantify the anisotropy of the distribution of interdomain orientations, we measured residual dipolar couplings (RDCs) from the two domains with multiple alignments. The N-terminal domain was directly aligned by a lanthanide ion and not influenced by interdomain motions, so it acted as a reference frame to achieve motional decoupling. We also applied {\it de novo} methods to extract spatial dynamic information from RDCs and represent interdomain motions as a continuous distribution on the 3D rotational space. Significant anisotropy was observed in the distribution, indicating the motion populates some interdomain orientations more than others. Statistical thermodynamic analysis of the observed orientational distribution suggests that it is among the energetically most favorable orientational distributions for binding to antibodies. Thus, the affinity is enhanced by a pre-posed distribution of interdomain orientations while maintaining the flexibility required for function.

The protocol described above can be applied to other biological systems in general. Protein molecule calmodulin and RNA molecule trans-activation response element (TAR) also have intensive interdomain motions with relative small intradomain dynamics. Their interdomain motions were studied using our method based on published RDC data. Our results were consistent with literature results in general. The differences could be due to previous studies' use of physical models, which contain assumptions about potential energy and thus introduced non-experimental information into the interpretations.

From traditional resource to global commodities: a comparison of Rhodiola species using NMR spectroscopy - metabolomics and HPTLC

The fast developing international trade of products based on traditional knowledge and their value chains has become an important aspect of the ethnopharmacological debate. The structure and diversity of value chains and their impact on the phytochemical composition of herbal medicinal products has been overlooked in the debate about quality problems in transnational trade. Different government policies and regulations governing trade in herbal medicinal products impact on such value chains. Medicinal Rhodiola species, including Rhodiola rosea L. and Rhodiola crenulata (Hook.f. & Thomson) H.Ohba, have been used widely in Europe and Asia as traditional herbal medicines with numerous claims for their therapeutic effects. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. We compare the phytochemical differences among Rhodiola raw materials available on the market to provide a practical method for Rhodiola authentication and the detection of potential adulterant compounds. Samples were collected from Europe and Asia and nuclear magnetic resonance spectroscopy coupled with multivariate analysis software and high performance thin layer chromatography techniques were used to analyse the samples. A method was developed to quantify the amount of adulterant species contained within mixtures. We compared the phytochemical composition of collected Rhodiola samples to authenticated samples. Rosavin and rosarin were mainly present in R. rosea whereas crenulatin was only present in R. crenulata. 30% of the Rhodiola samples purchased from the Chinese market were adulterated by other Rhodiola spp. Moreover, 7 % of the raw-material samples were not labelled satifactorily. The utilisation of both 1H-NMR and HPTLC methods provided an integrated analysis of the phytochemical differences and novel identification method for R. rosea and R. crenulata. Using 1H-NMR spectroscopy it was possible to quantify the presence of R. crenulata in admixtures with R. rosea. This quantitative technique could be used in the future to assess a variety of herbal drugs and products. This project also highlights the need to further study the links between producers and consumers in national and trans-national trade.

Tensor Completion for Multidimensional Inverse Problems with Applications to Magnetic Resonance Relaxometry

This thesis deals with tensor completion for the solution of multidimensional inverse problems. We study the problem of reconstructing an approximately low rank tensor from a small number of noisy linear measurements. New recovery guarantees, numerical algorithms, non-uniform sampling strategies, and parameter selection algorithms are developed. We derive a fixed point continuation algorithm for tensor completion and prove its convergence. A restricted isometry property (RIP) based tensor recovery guarantee is proved. Probabilistic recovery guarantees are obtained for sub-Gaussian measurement operators and for measurements obtained by non-uniform sampling from a Parseval tight frame. We show how tensor completion can be used to solve multidimensional inverse problems arising in NMR relaxometry. Algorithms are developed for regularization parameter selection, including accelerated k-fold cross-validation and generalized cross-validation. These methods are validated on experimental and simulated data. We also derive condition number estimates for nonnegative least squares problems. Tensor recovery promises to significantly accelerate N-dimensional NMR relaxometry and related experiments, enabling previously impractical experiments. Our methods could also be applied to other inverse problems arising in machine learning, image processing, signal processing, computer vision, and other fields.

Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and H-2 NMR

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, 2 H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S-mol) and dynamics (T-1) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a "transient site", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel. (C) 2007 Elsevier B.V. All rights reserved.

Nuclear Magnetic Resonance Spectroscopic and Computational Investigations of Chloroperoxidase Catalyzed Regio- and Enantio-Selective Transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.