Triple resonance, triple-quantum NMR studies of dipolar coupled spin systems

Multiple quantum-single quantum correlation experiments are employed for spectral simplification and determination of the relative signs of the couplings. In this study, we have demonstrated the excitation of three nuclei, triple quantum coherences and discussed the information obtainable from such experiments. The experiments have been carried out on doubly labeled acetonitrile and fluoroacetonitrile aligned in liquid crystalline media. The experiment is advantageous in providing many spectral parameters from a single experiment. The coherence pathways involved in the pulse sequence are described using product operators. (C) 2011 Elsevier Inc. All rights reserved.

TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below ∼25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.

Structural role of fluoride in aluminophosphate sol-gel glasses: High-resolution double-resonance NMR studies

The local structure of Na-Al-P-O-F glasses, prepared by a novel sol-gel route, was extensively investigated by advanced solid-state NMR techniques. Al-21{F-19} rotational echo double resonance (REDOR) results indicate that the F incorporated into aluminophosphate glass is preferentially bonded to octahedral Al units and results in a significant increase in the concentration of six-coordinated aluminum. The extent of Al-F and Al-O-P connectivities are quantified consistently by analyzing Al-27{P-31} and Al-21{F-19} REDOR NMR data. Two distinct types of fluorine species were identified and characterized by various F-19{Al-27}, F-19{Na-23}, and F-19{P-31} double resonance experiments, which were able to support peak assignments to bridging (Al-F-Al, -140 ppm) and terminal (Al-F, -170 ppm) units. On the basis of the detailed quantitative dipole-dipole coupling information obtained, a comprehensive structural model for these glasses is presented, detailing the structural speciation as a function of composition.

Analysis of Betaine and Choline Contents of Aleurone, Bran, and Flour Fractions of Wheat (Triticum aestivum L.) Using 1H Nuclear Magnetic Resonance (NMR) Spectroscopy

In conventional milling, the aleurone layer is combined with the bran fraction. Studies indicate that the bran fraction of wheat contains the majority of the phytonutrients betaine and choline, with relatively minor concentrations in the refined flour. This present study suggests that the wheat aleurone layer (Triticum aestivum L. cv. Tiger) contains the greatest concentration of both betaine and choline (1553.44 and 209.80 mg/100 g of sample, respectively). The bran fraction contained 866.94 and 101.95 mg/100 g of sample of betaine and choline, respectively, while the flour fraction contained 23.30 mg/100 g of sample (betaine) and 28.0 mg/100 g of sample (choline). The betaine content for
the bran was lower, and the choline content was higher compared to previous studies, although it is known that there is large variation in betaine and choline contents between wheat cultivars. The ratio of betaine/choline in the aleurone fraction was approximately 7:1; in the bran, the ratio was approximately 8:1; and in the flour fraction, the ratio was approximately 1:1. The study further
emphasizes the superior phytonutrient composition of the aleurone layer.
INTRODUCTION
Wheat is a valuable source of betaine, choline (1, 2), B
vitamins, vitamin E, and a number of minerals, including iron,
zinc, magnesium, and phosphorus (3). Epidemiological studies
indicate that whole-grain consumption is protective against
several chronic diseases (4-12). It has not been fully elucidated
how whole-grain cereals or specific fractions (13) exert their
protective effect, but it is thought to be due to their content of
several nutrients associated with the reduced risk of disease.
Conventionally, whole grain is separated during milling into
bran, germ, and flour (14). The nutrient composition of these
fractions differ markedly; refined wheat flour contains approximately
50% less vitamins and minerals than whole-grain
flour (

Structural role of fluoride in the ion-conducting glass system B(2)O(3)-PbO-LiF studied by single- and double-resonance NMR

The local structure of an ion-conducting glass with nominal composition 50B(2)O(3)-10PbO-40LiF has been investigated by complementary (7)Li, (11)B, (19)F, and (207)Pb single- and double-resonance experiments. The results give insight into the structural role of the lithium fluoride additive in borate glasses: (1) LiF is seen to actively participate in the network transformation process contributing to the conversion of three- into four-coordinate boron units, as shown by (11)B single-resonance as well as by (11)B{(19)F} and (19)F{(11)B} double-resonance experiments. (2) (19)F signal quantification experiments suggest substantial fluoride loss, presumably caused by formation of volatile BF(3). A part of the fluoride remains in the dopant role, possibly in the form of small LiF-like cluster domains, which serve as a mobile ion supply. (3) The extent of lithium-fluorine and lead-fluorine interactions has been characterized by (7)Li{(19)F} and (207)Pb{(19)F} REDOR and SEDOR experiments. On the basis of these results, a quantitative structural description of this system has been developed.

Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles

The 2H,13C,15N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30°C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The 13C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.

A nanoliter volume nuclear magnetic resonance (NMR) system using tunneling magneto-resistive (TMR) sensors to recognize biomolecules

The need to incorporate advanced engineering tools in biology, biochemistry and medicine is in great demand. Many of the existing instruments and tools are usually expensive and require special facilities.^ With the advent of nanotechnology in the past decade, new approaches to develop devices and tools have been generated by academia and industry. ^ One such technology, NMR spectroscopy, has been used by biochemists for more than 2 decades to study the molecular structure of chemical compounds. However, NMR spectrometers are very expensive and require special laboratory rooms for their proper operation. High magnetic fields with strengths in the order of several Tesla make these instruments unaffordable to most research groups.^ This doctoral research proposes a new technology to develop NMR spectrometers that can operate at field strengths of less than 0.5 Tesla using an inexpensive permanent magnet and spin dependent nanoscale magnetic devices. This portable NMR system is intended to analyze samples as small as a few nanoliters.^ The main problem to resolve when downscaling the variables is to obtain an NMR signal with high Signal-To-Noise-Ratio (SNR). A special Tunneling Magneto-Resistive (TMR) sensor design was developed to achieve this goal. The minimum specifications for each component of the proposed NMR system were established. A complete NMR system was designed based on these minimum requirements. The goat was always to find cost effective realistic components. The novel design of the NMR system uses technologies such as Direct Digital Synthesis (DDS), Digital Signal Processing (DSP) and a special Backpropagation Neural Network that finds the best match of the NMR spectrum. The system was designed, calculated and simulated with excellent results.^ In addition, a general method to design TMR Sensors was developed. The technique was automated and a computer program was written to help the designer perform this task interactively.^

Mg(2+) Ions Bind at the C-Terminal Region of Skeletal Muscle alpha-Tropomyosin

Tropomyosin (Tm) is a dimeric coiled-coil protein that polymerizes through head-to-tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may from sites for divalent cation binding. In a previous study, we showed that the Mg(2+)-induced increase in stability of the C-terminal half of Tin is sensitive to imitations near the C-terminus. In the present report, we study the interaction between Mg(2+) and full-length Tin and smaller fragments corresponding to the last 65 and 26 Tin residues. Although the smaller Tin peptide (Tm(259-284(W269))) is flexible and to large extent unstructured, the larger Tm(220-284(W269)) fragments forms a coiled coil in solution whose stability increases significantly in the presence of Mg(2+). NMR analysis shows thin Mg(2+) induces chemical shift perturbations in both Tm(220-284(W269)) and Tm(259-284(W269)) in the vicinity of His276, in which are located several negatively charged residues. (C) 2009 Wiley Periodicals, Inc. Biopolymers 91: 583-590, 2009.

Amino acid selective unlabeling for sequence specific resonance assignments in proteins

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective `unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly C-13/N-15 labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {(CO)-C-12 (i) -N-15 (i+1)}-filtered HSQC, which aids in linking the H-1(N)/N-15 resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i - 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to H-2 labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of N-14 at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

A study of water binding, mobility and dehydration in contact lens hydrogels using NMR

Hydrogel polymers are used for the manufacture of soft (or disposable) contact lenses worldwide today, but have a tendency to dehydrate on the eye. In vitro methods that can probe the potential for a given hydrogel polymer to dehydrate in vivo are much sought after. Nuclear magnetic resonance (NMR) has been shown to be effective in characterising water mobility and binding in similar systems (Barbieri, Quaglia et al., 1998, Larsen, Huff et al., 1990, Peschier, Bouwstra et al., 1993), predominantly through measurement of the spin-lattice relaxation time (T1), the spinspin relaxation time (T2) and the water diffusion coefficient (D). The aim of this work was to use NMR to quantify the molecular behaviour of water in a series of commercially available contact lens hydrogels, and relate these measurements to the binding and mobility of the water, and ultimately the potential for the hydrogel to dehydrate. As a preliminary study, in vitro evaporation rates were measured for a set of commercial contact lens hydrogels. Following this, comprehensive measurement of the temperature and water content dependencies of T1, T2 and D was performed for a series of commercial hydrogels that spanned the spectrum of equilibrium water content (EWC) and common compositions of contact lenses that are manufactured today. To quantify material differences, the data were then modelled based on theory that had been used for similar systems in the literature (Walker, Balmer et al., 1989, Hills, Takacs et al., 1989). The differences were related to differences in water binding and mobility. The evaporative results suggested that the EWC of the material was important in determining a material's potential to dehydrate in this way. Similarly, the NMR water self-diffusion coefficient was also found to be largely (if not wholly) determined by the WC. A specific binding model confirmed that the we was the dominant factor in determining the diffusive behaviour, but also suggested that subtle differences existed between the materials used, based on their equilibrium we (EWC). However, an alternative modified free volume model suggested that only the current water content of the material was important in determining the diffusive behaviour, and not the equilibrium water content. It was shown that T2 relaxation was dominated by chemical exchange between water and exchangeable polymer protons for materials that contained exchangeable polymer protons. The data was analysed using a proton exchange model, and the results were again reasonably correlated with EWC. Specifically, it was found that the average water mobility increased with increasing EWe approaching that of free water. The T1 relaxation was also shown to be reasonably well described by the same model. The main conclusion that can be drawn from this work is that the hydrogel EWe is an important parameter, which largely determines the behaviour of water in the gel. Higher EWe results in a hydrogel with water that behaves more like bulk water on average, or is less strongly 'bound' on average, compared with a lower EWe material. Based on the set of materials used, significant differences due to composition (for materials of the same or similar water content) could not be found. Similar studies could be used in the future to highlight hydrogels that deviate significantly from this 'average' behaviour, and may therefore have the least/greatest potential to dehydrate on the eye.

Diffusion-sensitive magnetic resonance spectroscopy and imaging in biomedical sciences

We present a mini-review of the development and contemporary applications of diffusion-sensitive nuclear magnetic resonance (NMR) techniques in biomedical sciences. Molecular diffusion is a fundamental physical phenomenon present in all biological systems. Due to the connection between experimentally measured diffusion metrics and the microscopic environment sensed by the diffusing molecules, diffusion measurements can be used for characterisation of molecular size, molecular binding and association, and the morphology of biological tissues. The emergence of magnetic resonance was instrumental to the development of biomedical applications of diffusion. We discuss the fundamental physical principles of diffusion NMR spectroscopy and diffusion MR imaging. The emphasis is placed on conceptual understanding, historical evolution and practical applications rather than complex technical details. Mathematical description of diffusion is presented to the extent that it is required for the basic understanding of the concepts. We present a wide range of spectroscopic and imaging applications of diffusion magnetic resonance, including colloidal drug delivery vehicles; protein association; characterisation of cell morphology; neural fibre tractography; cardiac imaging; and the imaging of load-bearing connective tissues. This paper is intended as an accessible introduction into the exciting and growing field of diffusion magnetic resonance.

High-Pressure Low-Temperature Locknut Cell For Both Electron-Paramagnetic-Res And Nmr-Studies To 10 Kilobars And 77-K

A locked high-pressure cell with working pressure range up to 10 kbars suitable for low-temperature studies to 77 K has been described. It can be used for both EPR and NMR studies of single crystals (and other solid samples). The high-pressure seal and all other aspects of the cell remain the same for either application. Only a change of the bottom plug is required for a switch from a nuclear-magnetic-resonance (NMR) to an electron-paramagnetic-resonance (EPR) experiment. Details of the procedure for the calibration of pressure inside the cell at various temperatures are discussed. The performance of the cell in EPR (Cr3+ion) and NMR (27Al nucleus) studies is reported.

1H and 19F NMR relaxation time studies in (NH4)2ZrF6 superionic conductor

1H and 19F spin-lattice relaxation times in polycrystalline diammonium hexafluorozirconate have been measured in the temperature range of 10–400 K to elucidate the molecular motion of both cation and anion. Interesting features such as translational diffusion at higher temperatures, molecular reorientational motion of both cation and anion groups at intermediate temperatures and quantum rotational tunneling of the ammonium group at lower temperatures have been observed. Nuclear magnetic resonance (NMR) relaxation time results correlate well with the NMR second moment and conductivity studies reported earlier.

NMR Spectroscopy of Multi-domain Proteins : Immunoglobulin-like Domains of Human Filamin A

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.