Solid-State NMR Literature Blog

Tuesday, January 05, 2010

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 9 December 2009
Peter T., While , Larry K., Forbes , Stuart, Crozier

Gradient coil temperature is an important concern in the design and construction of MRI scanners. Closely spaced gradient coil windings cause temperature hot spots within the system as a result of Ohmic heating associated with large current being driven through resistive material, and can strongly affect the performance of the coils. In this paper, a model is presented for predicting the spatial temperature distribution of a gradient coil, including the location and extent of temperature hot spots. Subsequently, a method is described for designing gradient coils with improved temperature distributions and reduced hot spot temperatures. Maximum temperature represents a non-linear constraint and a relaxed fixed point iteration routine is proposed to adjust coil windings iteratively to minimise this coil feature. Several examples are considered that assume different thermal material properties and cooling mechanisms for the gradient system. Coil winding solutions are obtained for all cases considered that display a considerable drop in hot spot temperature (>20%) when compared to standard minimum power gradient coils with equivalent gradient homogeneity, efficiency and inductance. The method is semi-analytical in nature and can be adapted easily to consider other non-linear constraints in the design of gradient coils or similar systems.

Homonuclear dipolar recoupling under ultra-fast magic-angle spinning: probing 19F19F proximities by solid-state NMR

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 16 December 2009
Qiang, Wang , Bingwen, Hu , Olivier, Lafon , Julien, Trébosc , Feng, Deng , and Jean-Paul Amoureux

We describe dipolar recoupling methods that accomplish, at high magic-angle spinning (MAS) frequencies, the excitation of double-quantum (DQ) coherences between spin-1/2 nuclei. We employ rotor-synchronized symmetry-based pulse sequences which are either γ-encoded or non-γ-encoded. The sensitivity and the robustness to both chemical-shift anisotropy and offset are examined. We also compare different techniques to avoid signal folding in the indirect dimension of two-dimensional double-quantum ↔ single-quantum (DQ−SQ) spectra. This comprehensive analysis results in the identification of satisfactory conditions for dipolar ¹⁹F−¹⁹F recoupling at high magnetic fields and high MAS frequencies. The utility of these recoupling methods is demonstrated with high-resolution DQ−SQ NMR spectra, which allow probing ¹⁹F−¹⁹F proximities in powered fluoroaluminates.

Intermolecular single-quantum coherence sequences for high-resolution NMR spectra in inhomogeneous fields

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 16 December 2009
Yuqing, Huang , Shuhui, Cai , Xi, Chen , Zhong, Chen

A new pulse sequence based on intermolecular single-quantum coherences (iSQCs) is proposed to obtain high-resolution NMR spectroscopy in inhomogeneous magnetic fields via fast 2D acquisition. Taking the intrinsic properties of iSQCs, the sequence is time-efficient with a narrow spectral width in the indirect dimension. It can recover useful information of chemical shifts, relative peak areas, J coupling constants, and multiplet patterns even when the field inhomogeneity is severe enough to erase almost all spectroscopic information. Moreover, good solvent suppression efficiency can be achieved by this sequence even with imperfect radio-frequency pulse flip angles. Spatially localized iSQC spectroscopy was performed on a sample packed with pig brain tissue and cucumber to show the feasibility of the sequence in in vivo magnetic resonance spectroscopy (MRS). This sequence may provide a promising way for the applications on in vivo and in situ high-resolution NMR spectroscopy.

Echo-time independent signal modulations for Strongly Coupled Systems in Triple Echo Localization schemes: An extension of S-PRESS editing

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 17 December 2009
Nils, Kickler , Giulio, Gambarota , Ralf, Mekle , Rolf, Gruetter , Robert, Mulkern

The double spin-echo point resolved spectroscopy sequence (PRESS) is a widely used method and standard in clinical MR spectroscopy. Existence of important J-modulations at constant echo times, depending on the temporal delays between the rf-pulses, have been demonstrated recently for strongly coupled spin systems and were exploited for difference editing, removing singlets from the spectrum (strong-coupling PRESS, S-PRESS). A drawback of this method for in vivo applications is that large signal modulations needed for difference editing occur only at relatively long echo times. In this work we demonstrate that, by simply adding a third refocusing pulse (3S-PRESS), difference editing becomes possible at substantially shorter echo times while, as applied to citrate, more favorable lineshapes can be obtained. For the example of an AB system an analytical description of the MR signal, obtained with this triple refocusing sequence (3S-PRESS), is provided.

On the applications of μr=-1 metamaterial lenses for magnetic resonance imaging

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 17 December 2009
Manuel J., Freire , Lukas, Jelinek , Ricardo, Marques , Mikhail, Lapine

In this work some possible applications of negative permeability magnetic metamaterial lenses for magnetic resonance imaging (MRI) are analyzed. It is shown that using magnetic metamaterials lenses it is possible to manipulate the spatial distribution of the radio-frequency (RF) field used in MR systems and, under some circumstances, improve the sensitivity of surface coils. Furthermore a collimation of the RF field, phenomenon that may find application in parallel imaging, is presented. MR images of real tissues are shown in order to prove the suitability of the theoretical analysis for practical applications.

L-Band Overhauser Dynamic Nuclear Polarization

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 21 December 2009
Sandra, Garcia , Jeffrey H., Walton , Brandon, Armstrong , Songi, Han , Michael J., McCarthy

We present the development of an Overhauser dynamic nuclear polarization (DNP) instrument at 0.04 T using 1.1 GHz (L-band) electron spin resonance frequencies (ESR) and 1.7 MHz ¹H nuclear magnetic resonance frequencies. Using this home-built DNP system, the electron–nucleus coupling factor of 4-oxo-TEMPO dissolved in water was determined as 0.39 ± 0.06 at 0.04 T. The higher coupling factor obtained at this field compared to higher magnetic fields, such as 0.35 T, directly translates to higher enhancement of the NMR signal and opens up a wider time scale window for observing water dynamics interacting with macromolecular systems, including proteins, polymers or lipid vesicles. The higher enhancements obtained will facilitate the observation of water dynamics at correlation times up to 10 ns, that corresponds to more than one order of magnitude slower dynamics than accessible at 0.35 T using X-band ESR frequencies.

Efficient Heteronuclear Dipolar Decoupling in Solid-State Nuclear Magnetic Resonance at Rotary Resonance Conditions

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 21 December 2009
Subhradip, Paul , Venus Singh, Mithu , Narayanan D., Kurur , P.K., Madhu

We introduce here a heteronuclear dipolar decoupling scheme in solid-state nuclear magnetic resonance that performs efficiently at the rotary-resonance conditions, where otherwise dipolar couplings are re-introduced. Results are shown proving the efficiency of this scheme at two magnetic fields under magic-angle spinning frequencies of 30 kHz and 20 kHz.

Homonuclear dipolar decoupling under fast MAS: Resolution patterns and simple optimization strategy

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 22 December 2009
Kanmi, Mao , Marek, Pruski

A simple method is shown for optimization of ¹H homonuclear dipolar decoupling at MAS rates exceeding 10 kHz. By monitoring the intensity of a spin-echo under the decoupling conditions, it is possible to optimize the amplitude of the RF magnetic field, the cycle time of the decoupling sequence and the resonance offset within minutes. As a result, the decoupling efficiency can be quickly and reliably fine-tuned without using a reference sample. The utility of this method has been confirmed by studying the resolution patterns for the supercycled PMLG scheme, which were found to be in excellent agreement with earlier theoretical predictions and verified in high-resolution 2D ¹H-¹H experiments.

Optimized Excitation Pulses for the Acquisition of Static NMR Powder Patterns from Half-Integer Quadrupolar Nuclei

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 23 December 2009
Luke A., O’Dell , Kristopher J., Harris , Robert W., Schurko

Various amplitude- and phase-modulated excitation pulses for the observation of static NMR powder patterns from half-integer quadrupolar nuclei have been generated using the optimal control routines implemented in SIMPSON 2.0. Such pulses are capable of both excitation of the central transition and signal enhancement by population transfer from the satellites. Enhancements in excess of 100% have been achieved for the central transition of the spin-3/2 ⁸⁷Rb nucleus compared with a selective π/2 pulse. These pulses are shown to be relatively insensitive to changes in RF power and transmitter offsets, and can achieve a more uniform signal enhancement than double frequency sweeps (DFS), resulting in more accurate spectral lineshapes. We also investigate the possibility of “calibration-free” optimized pulses for general use on half-integer quadrupoles with unknown interaction parameters. Such pulses could prove extremely useful for studying low abundance or insensitive nuclei for which experimental optimization of the DFS scheme may be difficult. We demonstrate that a pulse optimized for an arbitrary spin-3/2 system can function well on multiple samples, and can also excite the central transition of higher spin numbers, albeit with a smaller enhancement. The mechanism by which these optimized pulses achieve the signal enhancement is highly complex and, unlike DFS, involves a non-linear excitation of the satellite transition manifold, as well as the generation and manipulation of significant multiple-quantum coherences.

Benchmarking NMR experiments: a relational database of protein pulse sequences

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 23 December 2009
Russell R.P., Senthamarai , Ilya, Kuprov , Konstantin, Pervushin

Systematic benchmarking of multi-dimensional protein NMR experiments is a critical prerequisite for optimal allocation of NMR resources for structural analysis of challenging proteins, e.g. large proteins with limited solubility or proteins prone to aggregation. We propose a set of benchmarking parameters for essential protein NMR experiments organized into a lightweight (single XML file) relational database (RDB), which includes all the necessary auxiliaries (waveforms, decoupling sequences, calibration tables, setup algorithms and an RDB management system). The database is interfaced to the Spinach library (http://spindynamics.org), which enables accurate simulation and benchmarking of NMR experiments on large spin systems. A key feature is the ability to use a single user-specified spin system to simulate the majority of deposited solution state NMR experiments, thus providing the (hitherto unavailable) unified framework for pulse sequence evaluation. This development enables predicting relative sensitivity of deposited implementations of NMR experiments, thus providing a basis for comparison, optimization and, eventually, automation of NMR analysis. The benchmarking is demonstrated with two proteins, of 170 amino acids I domain of αXβ2 Integrin and 440 amino acids NS3 helicase.

Estimating Quadrupole Couplings of Amide Deuterons in Proteins from Direct Measurements of 2H Spin Relaxation Rates

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 24 December 2009
Devon, Sheppard , Vitali, Tugarinov

The measurements of longitudinal and transverse ²H spin relaxation rates of backbone amide deuterons (D^N) in the [U-¹³C,¹⁵N]-labeled protein ubiquitin show that the utility of amide deuterons as probes of backbone order in proteins is compromised by substantial variability of D^N quadrupolar coupling constants (QCC) from one amide site to another. However, using the dynamics parameters of ¹⁵N-²H bond vectors evaluated from ¹⁵N relaxation data, site-specific QCC values can be estimated directly from D^N R₁ and R₂ rates providing useful information on hydrogen bonding in proteins. In agreement with previous indirect scalar relaxation-based measurements, the D^N QCC values estimated directly from R₁ and R₂ ²H relaxation rates correlate with the inverse cube of the x-ray structure-derived hydrogen bond distances in ubiquitin: QCC = (232±2.3) + (118±17) Σ_i(cosα)r_i^-3 where r is the inter-nuclear hydrogen bond distance in ångströms, and α is the ND····O_i angle.

Nuclear acoustic resonance in fluids using piezoelectric nanoparticles

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 24 December 2009
J., Mende , N., Elmiladi , C., Höhl , K., Maier

The influence of acoustic radiation in the form of ultrasound (US) on the nuclear magnetic resonance (NMR) signal of liquids in the presence of piezo- and ferroelectric nanoparticles was investigated. The NMR resonances of ¹H and ²³Na were influenced by US with a frequency of ω_US = 18.26 MHz. For hydrogen, US with a frequency ω_US = ω₀ was used where ω₀ is the Larmor frequency of 18.26 MHz. For sodium, US with a frequency ω_US = 2ω_{0, Na} was used with ω_{0, Na} = 9.13 MHz. A detailed description of nanoparticle properties and sample preparation is given. The influence of US on the spin-lattice relaxation time T₁ was determined with an inversion recovery sequence for different concentrations of PZT. An elongation of T₁ of ¹H by 1.7% at a PZT concentration of 0.05% and an elongation of T₁ of ²³Na by 3% at a PZT concentration of 0.04% was observed. The elongation scales with the concentration of the PZT. An possible explanation of the effect of elongation is discussed.

A Practical Implementation of Cross-spectrum in Protein Backbone Resonance Assignment

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 24 December 2009
Kang, Chen , Frank, Delaglio , Nico, Tjandra

The concept of cross-spectrum is applied in protein NMR spectroscopy to assist in the backbone sequential resonance assignment. Cross-spectrum analysis is used routinely to reveal correlations in frequency domains as a means to reveal common features contained in multiple time series. Here the cross-spectrum between related NMR spectra, for example HNCO and HN(CA)CO, can be calculated with point-by-point multiplications along their common C’ carbon axis. In the resulting higher order cross-spectrum, an enhanced correlation signal occurs at every common i-1 carbon frequency allowing the amide proton H^N (and nitrogen N) resonances from residues i and i-1 to be identified. The cross-spectrum approach is demonstrated using 2D spectra H(N)CO, H(NCA)CO, H(NCO)CACB, and H(N)CACB measured on a ¹⁵N/¹³C double-labeled Ubiquitin sample. These 2D spectra are used to calculate two pseudo-3D cross-spectra, H_i-H_i-1-C’_i-1 and H_i-H_i-1-CA_i-1CB_i-1. We show using this approach, backbone resonances of H, C’, CA, and CB can be fully assigned without ambiguity. The cross-spectrum principle is expected to offer an easy, practical, and more quantitative approach for heteronuclear backbone resonance assignment.

Inter-residue carbonyl-carbonyl polarization transfer experiments in uniformly 13C,15N-labeled peptides and proteins

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 24 December 2009
Rafal, Janik , Emily, Ritz , Andrew, Gravelle , Lichi, Shi , Xiaohu, Peng , and Vladimir Ladizhansky

In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly ¹³C,¹⁵N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22 kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600 MHz. To match HORROR condition over all powder orientations, variable amplitude RF fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing interresidue long-range interresidue connectivities (e.g., (N[i]-CO[i-2])) are demonstrated on the model peptide N-Acetyl-Valine-Leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed.

Spectral Estimation of Irregularly Sampled Exponentially Decaying Signals with Applications to RF Spectroscopy

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2009
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 24 December 2009 Erik Gudmundson^a^,, Petre Stoica^a, Jian Li^b, Andreas Jakobsson^c, Michael D. Rowe^d, John A. S. Smith^d and Jun Ling^b

The problem of estimating the spectral content of exponentially decaying signals from a set of irregularly sampled data is of considerable interest in several applications, for example in various forms of radio frequency spectroscopy. In this paper, we propose a new nonparametric iterative adaptive approach that provides a solution to this estimation problem. As opposed to commonly used methods in the field, the damping coefficient, or linewidth, is explicitly modelled, which allows for an improved estimation performance. Numerical examples using both simulated data and data from NQR experiments illustrate the benefits of the proposed estimator as compared to currently available nonparametric methods.

Measurement of dipolar structure of 17O nuclear quadrupole resonance lines by three-frequency irradiation

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2010
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 4 January 2010
J., Seliger , V., Žagar

A modification of nuclear quadrupole double resonance with coupled multiplet is proposed which can be used for the measurement of the dipolar structure of the ¹⁷O nuclear quadrupole resonance lines in case of a strong ¹H-¹⁷O dipolar interaction. The technique is based on magnetic field cycling between a high magnetic field and zero magnetic field and on the simultaneous application of three rf magnetic fields with the frequencies that are close to the three ¹⁷O NQR frequencies ν_5/2-1/2 > ν_5/2-3/2 greater-or-equal, slanted

ν_3/2-1/2 during the time spent in zero static magnetic field. When the sum of the two lower irradiation frequencies ν₁+ν₂ is not equal to the highest irradiation frequencyν, the three-frequency irradiation increases the proton relaxation rate in zero magnetic field and consequently decreases the proton NMR signal at the end of the magnetic field cycle. The new technique is theoretically analyzed and compared to the single-frequency and two-frequency irradiation techniques. It is shown that the sensitivity of the new technique exceeds the sensitivity of the two-frequency irradiation technique. As a test of the new technique we measured the shape of the highest-frequency ¹⁷O NQR line in paraelectric KH₂PO₄.

Improving sensitivity and resolution of MQMAS spectra: a 45Sc-NMR case study of scandium sulphate pentahydrate

from ScienceDirect Publication: Journal of Magnetic Resonance

Publication year: 2010
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 4 January 2010
C. Vinod Chandran^a, Jérôme Cuny^b, Régis Gautier^b, Laurent Le Pollès^b, Chris J. Pickard^c and Thomas Bräuniger^a^,To efficiently obtain multiple-quantum magic-angle spinning (MQMAS) spectra of the nuclide ⁴⁵Sc (I=7/2), we have combined several previously suggested techniques to enhance the signal-to-noise ratio and to improve spectral resolution for the test sample, scandium sulphate pentahydrate (ScSPH). Whereas the ⁴⁵Sc-3QMAS spectrum of ScSPH does not offer sufficient resolution to clearly distinguish between the 3 scandium sites present in the crystal structure, these sites are well-resolved in the 5QMAS spectrum. The loss of sensitivity incurred by using MQMAS with 5Q coherence order is partly compensated for by using fast-amplitude modulated (FAM) sequences to improve the efficiency of both 5Q coherence excitation and conversion. Also, heteronuclear decoupling is employed to minimise dephasing of the ⁴⁵Sc signal during the 5Q evolution period due to dipolar couplings with the water protons in the ScSPH sample. Application of multi-pulse decoupling schemes such as TPPM and SPINAL results in improved sensitivity and resolution in the F₁ (isotropic) dimension of the 5QMAS spectrum, the best results being achieved with the recently suggested SW_f-TPPM sequence. By numerical fitting of the ⁴⁵Sc-NMR spectra of ScSPH from 3QMAS, 5QMAS and single-quantum MAS at magnetic fields B₀=9.4 T and 17.6 T, the isotropic chemical shift δ_iso, the quadrupolar coupling constant χ, and the asymmetry parameter η were obtained. Averaging over all experiments, the NMR parameters determined for the 3 scandium sites, designated (a), (b) and (c) are: δ_iso(a)=-15.5±0.5 ppm, χ(a)=5.60± 0.10 MHz, η(a)=0.06±0.05; δ_iso(b)=-12.9±0.5 ppm, χ(b)=4.50± 0.10 MHz, η(b)=1.00± 0.00; and δ_iso(c)=-4.7± 0.2 ppm, χ(c)=4.55± 0.05 MHz, η(c)=0.50±0.02. The NMR scandium species were assigned to the independent crystallographic sites by evaluating their experimental response to proton decoupling, and by density functional theory (DFT) calculations using the PAW and GIPAW approaches, in the following way: Sc(1) to (c), Sc(2) to (a), and Sc(3) to (b). The need to compute NMR parameters using an energy-optimised crystal structure is once again demonstrated.

Solid State Nuclear Magnetic Resonance

Multiple quadrupolar spin echoes in the La-filled skutterudite LaOs4As12

from ScienceDirect Publication: Solid State Nuclear Magnetic Resonance

Publication year: 2009
Source: Solid State Nuclear Magnetic Resonance, In Press, Accepted Manuscript, Available online 23 December 2009
B., Nowak , O., Żogał , Z., Henkie , M.B., Maple

We report experimental results of 139La pulse NMR studies in LaOs4As12. Measurements have been performed on a powder sample obtained from high quality single crystals. For the first time the pattern of quadrupole echoes for 139La nuclei (I=7/2) was obtained. All the allowed quadrupolar echoes expected for spin I=7/2 were observed at times t = (4/3)tau, (3/2) tau, (5/3) tau, 2 tau, (5/2) tau, 3 tau, 4 tau. The presence of quadrupolar echoes is the fingerprint of the deviation from perfect cubic symmetry of the structure and can be used as a simple and fast test of the sample quality.

Saving transverse magnetization

from ScienceDirect Publication: Solid State Nuclear Magnetic Resonance

Publication year: 2009
Source: Solid State Nuclear Magnetic Resonance, In Press, Accepted Manuscript, Available online 21 December 2009
Takeshi, Kobayashi , Joseph A., DiVerdi , Rebecca A., Faulkner , Gary E., Maciel

A magnetization storage sequence, ALT-1 (alternating longitudinal and transverse components), is reported. The ALT-1 sequence is a hybrid of two types of storage sequences, the Carr–Purcell type and store-and-restore sequences. During incremental storage periods within the ALT-1 sequence, essentially half of the initially transverse magnetization is stored along the z-axis and the other half is prolonged by an echo-generating pulse. The portions of initial magnetization that are stored as longitudinal components or transverse components are alternated by a π/2 pulse during the cycle. Both transverse components of the initial magnetization are treated the same in the ALT-1 sequence and orientational (phase) information of the initial magnetization is kept during the storage period. The ALT-1 sequence can preserve magnetization more effectively than a published class of modified Carr–Purcell type sequences, because essentially half of the magnetization during incremental storage periods is not subjected to relaxation from T₂ effects.

Magnetic resonance tensors in uracil: Calculation of 13C, 15N, 17O NMR chemical shifts, 17O and 14N electric field gradients and measurement of 13C and 15N chemical shifts

from ScienceDirect Publication: Solid State Nuclear Magnetic Resonance

Publication year: 2009
Source: Solid State Nuclear Magnetic Resonance, In Press, Accepted Manuscript, Available online 16 December 2009
Saeed K., Amini , Hoora, Shaghaghi , Alex D., Bain , Ammar, Chabok , Mohsen, Tafazzoli

The experimental ¹³C NMR chemical shift components of uracil in the solid state are reported for the first time (to our knowledge), as well as newer data for the ¹⁵N nuclei. These experimental values are supported by extensive calculated data of the ¹³C, ¹⁵N and ¹⁷O chemical shielding and ¹⁷O and ¹⁴N electric field gradient (EFG) tensors. In the crystal, uracil forms a number of strong and weak hydrogen bonds, and the effect of these on the ¹³C and ¹⁵N chemical shift tensors is studied. This powerful combination of the structural methods and theoretical calculations gives a very detailed view of the strong and weak hydrogen bond formation by this molecule. Good calculated results for the optimized cluster in most cases (except for the EFG values of the ¹⁴N3 and ¹⁷O4 nuclei) certify the accuracy of our optimized coordinates for the hydrogen nuclei. Our reported RMSD values for the calculated chemical shielding and EFG tensors are smaller than those reported previously. In the optimized cluster the 6-311+G** basis set is the optimal one in the chemical shielding and EFG calculations, except for the EFG calculations of the oxygen nuclei, in which the 6-31+G** basis set is the optimal one. The optimal method for the chemical shielding and EFG calculations of the oxygen and nitrogen nuclei is the PW91PW91 method, while for the chemical shielding calculations of the ¹³C nuclei the B3LYP method gives the best results.

Monday, December 14, 2009

J. Chem. Phys. 131, 224516 (2009); doi:10.1063/1.3266422

Spatial/spectral encoding of the spin interactions in ultrafast multidimensional NMR
Yoav Shrot and Lucio Frydman

Abstract:
Two-dimensionalnuclear magnetic resonance (2D NMR) spectroscopy provides the means toextract diverse physical, chemical, and biological information at an atomiclevel. Conventional sampling schemes, however, may result in relatively long2D experiments; this has stimulated the search for alternative, rapidacquisition schemes. Among the strategies that have been recently proposedfor achieving this counts an “ultrafast” approach, relying on thespatial encoding of the indirect domain evolution to provide arbitraryspectra within a single scan. A common feature of allspatial encoding schemes hitherto described is their uniform encoding ofa continuous bandwidth of indirect-domain frequencies, regardless of the chemicalsites' spectral distribution within it. These very general conditions, however,are often associated with a number of tradeoffs and compromisesin the spectral widths and resolutions that can be achievedfor both the direct and indirect domains. This paper proposesa different strategy for single-scan acquisition of 2D spectra, whichperforms an optimal encoding by employing a priori information regardingthe positions of NMR resonances along the indirect domain. Wedenote this as “spatial/spectral encoding”; the underlying principles of thisnew approach, together with experimental results based on uni- andmultidimensional rf pulse schemes, are presented. ©2009 American Institute of Physics

J. Am. Chem. Soc., 2009, 131 (49), pp 17972–17979

NMR Structure in a Membrane Environment Reveals Putative Amyloidogenic Regions of the SEVI Precursor Peptide PAP248−286
Ravi P. R. Nanga†, Jeffrey R. Brender†‡, Subramanian Vivekanandan†‡, Nataliya Popovych†‡ and Ayyalusamy Ramamoorthy*†

Abstract
Semen is the main vector for HIV transmission worldwide. Recently, a peptide fragment (PAP248−286) has been isolated from seminal fluid that dramatically enhances HIV infectivity by up to 4−5 orders of magnitude. PAP248−286 appears to enhance HIV infection by forming amyloid fibers known as SEVI, which are believed to enhance the attachment of the virus by bridging interactions between virion and host-cell membranes. We have solved the atomic-level resolution structure of the SEVI precursor PAP248−286 using NMR spectroscopy in SDS micelles, which serve as a model membrane system. PAP248−286, which does not disrupt membranes like most amyloid proteins, binds superficially to the surface of the micelle, in contrast to other membrane-disruptive amyloid peptides that generally penetrate into the core of the membrane. The structure of PAP248−286 is unlike most amyloid peptides in that PAP248−286 is mostly disordered when bound to the surface of the micelle, as opposed to the α-helical structures typically found of most amyloid proteins. The highly disordered nature of the SEVI peptide may explain the unique ability of SEVI amyloid fibers to enhance HIV infection as partially disordered amyloid fibers will have a greater capture radius for the virus than compact amyloid fibers. Two regions of nascent structure (an α-helix from V262−H270 and a dynamic α/310 helix from S279−L283) match the prediction of highly amyloidogenic sequences and may serve as nuclei for aggregation and amyloid fibril formation. The structure presented here can be used for the rational design of mutagenesis studies on SEVI amyloid formation and viral infection enhancement.

J. Am. Chem. Soc., 2009, 131 (49), pp 17908–17918

Quantitative Description of Backbone Conformational Sampling of Unfolded Proteins at Amino Acid Resolution from NMR Residual Dipolar Couplings
Gabrielle Nodet†, Loc Salmon†, Valry Ozenne†, Sebastian Meier‡, Malene Ringkjøbing Jensen† and Martin Blackledge*†

Abstract
An atomic resolution characterization of the structural properties of unfolded proteins that explicitly invokes the highly dynamic nature of the unfolded state will be extremely important for the development of a quantitative understanding of the thermodynamic basis of protein folding and stability. Here we develop a novel approach using residual dipolar couplings (RDCs) from unfolded proteins to determine conformational behavior on an amino acid specific basis. Conformational sampling is described in terms of ensembles of structures selected from a large pool of conformers. We test this approach, using extensive simulation, to determine how well the fitting of RDCs to reduced conformational ensembles containing few copies of the molecule can correctly reproduce the backbone conformational behavior of the protein. Having established approaches that allow accurate mapping of backbone dihedral angle conformational space from RDCs, we apply these methods to obtain an amino acid specific description of ubiquitin denatured in 8 M urea at pH 2.5. Cross-validation of data not employed in the fit verifies that an ensemble size of 200 structures is appropriate to characterize the highly fluctuating backbone. This approach allows us to identify local conformational sampling properties of urea-unfolded ubiquitin, which shows that the backbone sampling of certain types of charged or polar amino acids, in particular threonine, glutamic acid, and arginine, is affected more strongly by urea binding than amino acids with hydrophobic side chains. In general, the approach presented here establishes robust procedures for the study of all denatured and intrinsically disordered states.

J. Am. Chem. Soc., 2009, 131 (49), pp 17756–17758

Widening the View on Dispersant−Pigment Interactions in Colloidal Dispersions with Saturation Transfer Difference NMR Spectroscopy
Agnieszka Szczygiel†, Leo Timmermans‡, Bernd Fritzinger† and Jos C. Martins*†

Abstract
The application of Saturation Transfer Difference (STD) NMR spectroscopy for the characterization of dispersant particle interactions is introduced. STD NMR has hitherto been applied, with great success, to the characterization of ligand−protein interactions and is currently a standard tool in biomolecular NMR spectroscopy. Nevertheless, the STD NMR technique has so far not yet crossed the boundaries of the biomolecular field. Here, we demonstrate that in spite of clear differences between a protein binding site and the surface of a pigment nanoparticle, the latter can also be subjected to STD NMR analysis, allowing us to detect (screen for) binding ligands, discriminate ligand from nonligand, and obtain information on the binding epitope. The approach should be generally applicable as long as the nanoparticle is comprised of a dense network of hydrogens, implicating almost all organic molecular nanocrystals. Thus it provides a novel investigative tool for the study of dispersions that is highly complementary to existing ones.

Friday, December 11, 2009

J. Am. Chem. Soc., 2009, 131 (48), pp 17576–17582

Ternary Protein Complex of Ferredoxin, Ferredoxin:Thioredoxin Reductase, and Thioredoxin Studied by Paramagnetic NMR Spectroscopy
Xingfu Xu†, Peter Schrmann‡, Jung-Sung Chung§, Mathias A. S. Hass†, Sung-Kun Kim§, Masakazu Hirasawa§, Jatindra N. Tripathy, David B. Knaff§ and Marcellus Ubbink*†

Abstract
In oxygenic photosynthetic cells, carbon metabolism is regulated by a light-dependent redox signaling pathway through which the light signal is transmitted in the form of electrons via a redox chain comprising ferredoxin (Fd), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx). Trx affects the activity of a variety of enzymes via dithiol oxidation and reduction reactions. FTR reduces an intramolecular disulfide bridge of Trx, and Trx reduction involves a transient cross-link with FTR. NMR spectroscopy was used to investigate the interaction of Fd, FTR, and an m-type Trx. NMR titration experiments indicate that FTR uses distinct sites to bind Fd and Trx simultaneously to form a noncovalent ternary complex. The orientation of Trx-m relative to FTR was determined from the intermolecular paramagnetic broadening caused by the [4Fe-4S] cluster of FTR. Two models of the noncovalent binary complex of FTR/Trx-m based on the paramagnetic distance restraints were obtained. The models suggest that either a modest or major rotational movement of Trx must take place when the noncovalent binary complex proceeds to the covalent complex. This study demonstrates the complementarity of paramagnetic NMR and X-ray diffraction of crystals in the elucidation of dynamics in a transient protein complex.

J. Am. Chem. Soc., 2009, 131 (47), pp 17064–17065

Time-Resolved Dehydration-Induced Structural Changes in an Intact Bovine Cortical Bone Revealed by Solid-State NMR Spectroscopy
Peizhi Zhu†, Jiadi Xu†‡, Nadder Sahar§, Michael D. Morris*†§, David H. Kohn§ and Ayyalusamy Ramamoorthy*†‡

Abstract
Understanding the structure and structural changes of bone, a highly heterogeneous material with a complex hierarchical architecture, continues to be a significant challenge even for high-resolution solid-state NMR spectroscopy. While it is known that dehydration affects mechanical properties of bone by decreasing its strength and toughness, the underlying structural mechanism at the atomic level is unknown. Solid-state NMR spectroscopy, controlled dehydration, and H/D exchange were used for the first time to reveal the structural changes of an intact piece of bovine cortical bone. 1H spectra were used to monitor the dehydration of the bone inside the rotor, and high-resolution 13C chemical shift spectra obtained under magic-angle spinning were used evaluate the dehydration-induced conformational changes in the bone. The experiments revealed the slow denaturation of collagen due to dehydration while the trans-Xaa-Pro conformation in collagen remained unchanged. Our results suggest that glycosaminoglycans in the collagen fiber and mineral interface may chelate with a Ca2+ ion present on the surface of the mineral through sulfate or carboxylate groups. These results provide insights into the role of water molecules in the bone structure and shed light on the relationship between the structure and mechanics of bone.

J. Am. Chem. Soc., 2009, 131 (47), pp 17054–17055

Double-Nucleus Enhanced Recoupling for Efficient 13C MAS NMR Correlation Spectroscopy of Perdeuterated Proteins
Umit Akbey, Hartmut Oschkinat and Barth-Jan van Rossum*

Abstract
The use of both proton and deuterium dipolar coupling networks to obtain efficient 13C magic-angle-spinning NMR correlation spectroscopy is introduced. This new strategy aims to improve the efficiency of 13C spin diffusion in perdeuterated proteins. The method is called double-nucleus enhanced recoupling (DONER), and it provides significantly improved transfer efficiency for carbon spin diffusion at low proton density. The Cα cross-peak intensity in the DONER experiment is 3 and 5 times stronger than those in conventional radio-frequency-assisted diffusion (RAD) and proton-driven spin diffusion (PDSD) experiments, respectively. Remarkably, the full cross-peak pattern for the aliphatic region of an extensively perdeuterated SH3 protein sample can be obtained using the DONER approach with direct 13C excitation.

Wednesday, December 09, 2009

J. Am. Chem. Soc., 2009, 131 (44), pp 16257–16265

Selective Characterization of Microsecond Motions in Proteins by NMR Relaxation
D. Flemming Hansen*†, Haniqiao Feng‡, Zheng Zhou‡, Yawen Bai‡ and Lewis E. Kay*†

Abstract
The three-dimensional structures of macromolecules fluctuate over a wide range of time-scales. Separating the individual dynamic processes according to frequency is of importance in relating protein motions to biological function and stability. We present here a general NMR method for the specific characterization of microsecond motions at backbone positions in proteins even in the presence of other dynamics such as large-amplitude nanosecond motions and millisecond chemical exchange processes. The method is based on measurement of relaxation rates of four bilinear coherences and relies on the ability of strong continuous radio frequency fields to quench millisecond chemical exchange. The utility of the methodology is demonstrated and validated through two specific examples focusing on the thermo-stable proteins, ubiquitin and protein L, where it is found that small-amplitude microsecond dynamics are more pervasive than previously thought. Specifically, these motions are localized to α helices, loop regions, and regions along the rim of β sheets in both of the proteins examined. A third example focuses on a 28 kDa ternary complex of the chaperone Chz1 and the histones H2A.Z/H2B, where it is established that pervasive microsecond motions are localized to a region of the chaperone that is important for stabilizing the complex. It is further shown that these motions can be well separated from extensive millisecond dynamics that are also present and that derive from exchange of Chz1 between bound and free states. The methodology is straightforward to implement, and data recorded at only a single static magnetic field are required.

J. Am. Chem. Soc., 2009, 131 (44), pp 16014–16015

Proton NMR of 15N-Choline Metabolites Enhanced by Dynamic Nuclear Polarization
Riddhiman Sarkar†, Arnaud Comment‡§, Paul R. Vasos†, Sami Jannin, Rolf Gruetter‡§, Geoffrey Bodenhausen†, Hlne Hall, Deniz Kirik# and Vladimir P. Denisov#

Abstract
Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in 15N-labeled choline up to 3.3% by irradiating the 94 GHz electron spin resonance of admixed TEMPO nitroxide radicals in a magnetic field of 3.35 T during ca. 3 h at 1.2 K. The sample was subsequently transferred to a high-resolution magnet, and the enhanced polarization was converted from 15N to methyl- and methylene protons, using the small 2,3J(1H,15N) couplings in choline. The room-temperature lifetime of nitrogen polarization in choline, T1(15N) ≈ 200 s, could be considerably increased by partial deuteration of the molecule. This procedure enables studies of choline metabolites in vitro and in vivo using DNP-enhanced proton NMR.

J. Am. Chem. Soc., 2009, 131 (44), pp 15994–15995

Deconvolution of Complex NMR Spectra in Small Molecules by Multi Frequency Homonuclear Decoupling (MDEC)
Ana Paula D. M. Espindola†, Ronald Crouch‡, John R. DeBergh†, Joseph M. Ready† and John B. MacMillan*†

Abstract
A new technique to deconvolute complex 1H NMR spectra of small molecules has been developed that utilizes shape selective pulses to simultaneously decouple multiple protons. A limitation in the assignment of the relative configuration of small molecules is the ability to accurately obtain coupling constants. Other methods such as the E.COSY and the 2D J-resolved are available to obtain complicated coupling constants; the multiple homonuclear decoupling method (MDEC) described is a rapid and simple technique. Three examples of increasing spectral complexity, menthol, cholesteryl acetate and a C16 fatty acid, demonstrate the utility of the technique. Increasing the experimental utility, the single pulse MDEC experiment can be incorporated in other 1D experiments, such as a 1D-TOCSY to solve specific problems.

J. Am. Chem. Soc., 2009, 131 (44), pp 15982–15983

Monitoring of Biological One-Electron Reduction by 19F NMR Using Hypoxia Selective Activation of an 19F-Labeled Indolequinone Derivative
Kazuhito Tanabe*†, Hiroshi Harada‡§, Michiko Narazaki, Kazuo Tanaka, Kenichi Inafuku, Hirokazu Komatsu†, Takeo Ito†, Hisatsugu Yamada†§, Yoshiki Chujo, Tetsuya Matsuda, Masahiro Hiraoka‡§ and Sei-ichi Nishimoto*†

Abstract
Biological reduction of fluorine-labeled indolequinone derivative (IQ-F) was characterized by 19F NMR for quantitative molecular understanding. The chemical shift change in 19F NMR allowed monitoring of the enzymatic reduction of IQ-F. Upon hypoxic treatment of IQ-F with NADPH:cytochrome P450 reductase, IQ-F was activated via catalytic one-electron reduction to release nonafluoro-tert-butyl alcohol (F-OH), while the formation of F-OH was significantly suppressed under aerobic conditions. Similar hypoxia-selective reduction of IQ-F occurred within A549 cells, which expresses NADPH:cytochrome P450 reductase. The kinetic analysis was also performed to propose a reaction mechanism. The molecular oxygen slightly prevents the binding of IQ-F to reductase, while the rate of net reaction was decreased due to oxidation of a semiquinone anion radical intermediate generated by one-electron reduction of IQ-F. The disappearance of IQ-F and appearance of F-OH were imaged by 19F fast spin echo, thus visualizing the hypoxia-selective reduction of IQ-F by means of MR imaging.

J. Am. Chem. Soc., 2009, 131 (44), pp 15970–15971

Higher Sensitivity through Selective 13C Excitation in Solid-State NMR Spectroscopy
Jakob J. Lopez*, Christoph Kaiser, Sam Asami and Clemens Glaubitz*

Abstract
A notable drawback of NMR spectroscopy is its inherently low sensitivity: 95% of the measuring time consists solely of idle delays during which nuclei regain their Boltzmann equilibrium. Here, a strategy for solid-state 13C NMR experiments is presented that allows the user to acquire spectra in time periods that are notably shorter than previously necessary. Experiments that are band-selective in nature may utilize the cooling potential of unperturbed nuclei to lower the spin temperature of their excited neighbors. As we demonstrate, it becomes possible to replace the recycle delay in a series of scans by a time period during which proton-driven spin diffusion causes relaxation enhancement by a lower spin temperature of adjacent spins (RELOAD). Typically, a duration of 200 ms suffices for this step, and for 1D 13C NMR experiments, it is shown that the omission of recycle delays (typically of 2 s length) reduces the measuring time substantially. RELOAD is applied to 2D homonuclear 13C NMR experiments, and it is demonstrated that for experiments in which correlations between 13C backbone atoms are detected, the measurement time is reduced by a factor of 10 through a time-saving combination of a smaller number of increments in the indirect dimension and RELOAD.

J. Am. Chem. Soc., 2009, 131 (44), pp 15968–15969

Large Protein Complexes with Extreme Rotational Correlation Times Investigated in Solution by Magic-Angle-Spinning NMR Spectroscopy
Andi Mainz†, Stefan Jehle†, Barth J. van Rossum†, Hartmut Oschkinat† and Bernd Reif*†‡

Abstract
We show that large protein complexes can be investigated in solution using magic-angle-spinning (MAS) NMR spectroscopy without the need for sample crystallization or precipitation. In order to efficiently average anisotropic interactions with MAS, the rotational diffusion of the molecule has to be suppressed. This can be readily achieved by lowering the sample temperature and by adding glycerol to the protein solution. The approach is demonstrated using the human small heat shock protein (sHSP) αB-Crystallin, which forms oligomeric assemblies of 600 kDa. We suggest this scheme as an approach for overcoming size limitations imposed by overall tumbling in solution-state NMR investigations of large protein complexes.

Wednesday, December 02, 2009

J. Am. Chem. Soc., 2009, 131 (43), pp 15761–15768

Evaluation of Parameters Critical for Observing Nucleic Acids Inside Living Xenopus laevis Oocytes by In-Cell NMR Spectroscopy
Robert Hnsel†‡, Silvie Foldynov-Trantrkov§, Frank Lhr†‡, Janina Buck‡, Eva Bongartz†, Ernst Bamberg†, Harald Schwalbe‡, Volker Dtsch*†‡ and Luk Trantrek*§#

Abstract
In-cell NMR spectroscopy of proteins in different cellular environments is a well-established technique that, however, has not been applied to nucleic acids so far. Here, we show that isotopically labeled DNA and RNA can be observed inside the eukaryotic environment of Xenopus laevis oocytes by in-cell NMR spectroscopy. One limiting factor for the observation of nucleic acids in Xenopus oocytes is their reduced stability. We demonstrate that chemical modification of DNA and RNA can protect them from degradation and can significantly enhance their lifetime. Finally, we show that the imino region of the NMR spectrum is devoid of any oocyte background signals enabling the detection even of isotopically nonlabeled molecules.

J. Am. Chem. Soc., 2009, 131 (43), pp 15596–15597

Chemical Labeling Strategy with (R)- and (S)-Trifluoromethylalanine for Solid State 19F NMR Analysis of Peptaibols in Membranes
Daniel Maisch†, Parvesh Wadhwani‡, Sergii Afonin‡, Christoph Bttcher§, Beate Koksch§ and Anne S. Ulrich*†‡

Abstract
Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints θ by solid state 19F NMR. The structure of the membrane-perturbing antibiotic alamethicin in DMPC bilayers was analyzed in terms of two angles τ and ρ from six such constraints, showing that the N-terminus (up to a kink at Pro14) is folded as an α-helix, tilted away from the membrane normal by 8°, and assembled as an oligomer. The new 19F NMR label CF3-Ala has thus been demonstrated to be highly sensitive, virtually unperturbing, and ideally suited to characterize peptaibols in membranes.

Monday, October 26, 2009

Cryst. Growth Des., Article ASAP

Solid-State NMR, X-ray Diffraction, and Thermoanalytical Studies Towards the Identification, Isolation, and Structural Characterization of Polymorphs in Natural Bile Acids
Nonappa, Manu Lahtinen, Satu Ikonen, Erkki Kolehmainen* and Reijo Kauppinen

AbstractCombined solid-state NMR, powder, and single crystal X-ray diffraction, as well as thermoanalytical studies were performed towards the identification, isolation, and structural characterization of polymorphs present in commercial samples of six natural bile acids. The 13C{1H} cross-polarization (CP) magic angle spinning (MAS) NMR technique was used as a primary tool to identify the mixture of polymorphs present in the natural bile acids obtained from commercial sources. A detailed study including selective crystallization of each polymorphic form of the bile acids in different solvents and mixture of solvents was carried out, and their spectral patterns were compared with the mode of packing and number of molecules present in an asymmetric unit. These studies were strongly supported by other complementary techniques such as powder/single crystal X-ray diffraction and differential scanning calorimetric (DSC) experiments. While a few single crystal X-ray structures are solved in order to confirm the correct polymorphic form, most of the powder X-ray diffraction data were compared with the simulated X-ray patterns obtained from previously reported single crystal structures. Detailed analyses using multiple experimental techniques provided useful insights towards the mode of packing which is responsible for unique spectral patterns in the solid state.

Friday, October 23, 2009

J. Am. Chem. Soc., Article ASAP

Deuterium Spin Probes of Backbone Order in Proteins: 2H NMR Relaxation Study of Deuterated Carbon α Sites
Devon Sheppard‡, Da-Wei Li§, Rafael Brschweiler§ and Vitali Tugarinov*‡

Abstract
2H spin relaxation NMR experiments to study the dynamics of deuterated backbone α-positions, Dα, are developed. To date, solution-state 2H relaxation measurements in proteins have been confined to side-chain deuterons—primarily 13CH2D or 13CHD2 methyl groups. It is shown that quantification of 2H relaxation rates at Dα backbone positions and the derivation of associated order parameters of Cα−Dα bond vector motions in small [U-15N,13C,2H]-labeled proteins is feasible with reasonable accuracy. The utility of the developed methodology is demonstrated on a pair of proteins—ubiquitin (8.5 kDa) at 10, 27, and 40 °C, and a variant of GB1 (6.5 kDa) at 22 °C. In both proteins, the Dα-derived parameters of the global rotational diffusion tensor are in good agreement with those obtained from 15N relaxation rates. Semiquantitative solution-state NMR measurements yield an average value of the quadrupolar coupling constant, QCC, for Dα sites in proteins equal to 174 kHz. Using a uniform value of QCC for all Dα sites, we show that Cα−Dα bond vectors are motionally distinct from the backbone amide N−H bond vectors, with 2H-derived squared order parameters of Cα−Dα bond vector motions, S2CαDα, on average slightly higher than their N−H amides counterparts, S2NH. For ubiquitin, the 2H-derived backbone mobility compares well with that found in a 1-μs molecular dynamics simulation.

J. Am. Chem. Soc., 2009, 131 (42), pp 15086–15087

1H and 13C Dynamic Nuclear Polarization in Aqueous Solution with a Two-Field (0.35 T/14 T) Shuttle DNP Spectrometer
Marcel Reese†, Maria-Teresa Trke†, Igor Tkach†, Giacomo Parigi‡, Claudio Luchinat‡, Thorsten Marquardsen§, Andreas Tavernier§, Peter Hfer§, Frank Engelke§, Christian Griesinger*† and Marina Bennati*†

Abstract
Dynamic nuclear polarization (DNP) permits increasing the NMR signal of nuclei by pumping the electronic spin transitions of paramagnetic centers nearby. This method is emerging as a powerful tool to increase the inherent sensitivity of NMR in structural biology aiming at detection of macromolecules. In aqueous solution, additional technical issues associated with the penetration of microwaves in water and heating effects aggravate the performance of the experiment. To examine the feasibility of low-field (9.7 GHz/0.35 T) DNP in high resolution NMR, we have constructed the prototype of a two-field shuttle DNP spectrometer that polarizes nuclei at 9.7 GHz/0.35 T and detects the NMR spectrum at 14 T. We report our first 1H and 13C DNP enhancements with this spectrometer. Effective enhancements up to 15 were observed for small molecules at 1H 600 MHz/14 T as compared to the Boltzmann signal. The results provide a proof of principle for the feasibility of a shuttle DNP experiment and open up perspectives for the application potential of this method in solution NMR.

J. Am. Chem. Soc., 2009, 131 (36), pp 12949–12959

Design and NMR-Based Screening of LEF, a Library of Chemical Fragments with Different Local Environment of Fluorine
Anna Vulpetti*§, Ulrich Hommel§, Gregory Landrum§, Richard Lewis§ and Claudio Dalvit*§#

Abstract
A novel strategy for the design of a fluorinated fragment library that takes into account the local environment of fluorine is described. The procedure, based on a fluorine fingerprints descriptor, and the criteria used in the design, selection, and construction of the library are presented. The library, named LEF (Local Environment of Fluorine), combined with 19F NMR ligand-based screening experiments represents an efficient and sensitive approach for the initial fragment identification within a fragment-based drug discovery project and for probing the presence of fluorophilic protein environments. Proper setup of the method, according to described theoretical simulations, allows the detection of very weak-affinity ligands and the detection of multiple ligands present within the same tested mixture, thus capturing all the potential fragments interacting with the receptor. These NMR hits are then used in the FAXS experiments for the fragment optimization process and for the follow-up screening aimed at identifying other chemical scaffolds relevant for the binding to the receptor

Thursday, October 22, 2009

Cryst. Growth Des., 2009, 9 (10), pp 4281–4288

Effects of Crystal Growth and Polymorphism of Triacylglycerols on NMR Relaxation Parameters. 2. Study of a Tricaprin−Tristearin Mixture
Matthieu Adam-Berret†‡§, Alain Riaublanc‡ and Franois Mariette*†§

Abstract
Being able to determine the physical properties of fats such as polymorphism and crystal size is very important for the food industry. After a relationship was observed between spin−lattice relaxation time and crystal size in a solid−liquid mixture of triacylglycerols, the effects of polymorphism and crystal size were investigated by low-field NMR relaxation and powder X-ray diffraction on three binary mixtures of tricaprin and tristearin in the solid state. Second moment (M2) was proven to be only sensitive to polymorphism. Its measurements permitted the quantification of polymorphic forms in a binary mixture, with a model based on M2 of the pure components. As for the spin−lattice relaxation time (T1), it was proven to be only sensitive to crystal size and not to polymorphism. Quantification was not possible with T1 measurements, but information on the pattern of the crystal thickness distribution was obtained using the maximization entropy method algorithm. The determination of polymorphism was thus possible because of the difference in size between the α and β triacylglycerol crystals. Finally, a phase diagram mainly based on NMR data was constructed for the tricaprin/tristearin system.

Cryst. Growth Des., 2009, 9 (10), pp 4273–4280

Effects of Crystal Growth and Polymorphism of Triacylglycerols on NMR Relaxation Parameters. 1. Evidence of a Relationship between Crystal Size and Spin−Lattice Relaxation Time
Matthieu Adam-Berret†‡§, Alain Riaublanc‡, Corinne Rondeau-Mouro‡ and Franois Mariette*‡§

Abstract
Fat crystal networks confer their physical properties on fat-containing products. They are characterized by the solid fat content (SFC) and the design of the crystals, that is, their polymorphism and their size. Different techniques such as NMR, differential scanning calorimetry, and X-ray diffraction (XRD) are used to determine these parameters. Low-field NMR, the reference method for evaluation of SFC, has been proven to allow the determination of polymorphism through spin−lattice relaxation time (T1) and second moment (M2) measurements. However, this technique could provide more information on the system. On the basis of the effects of supercooling on the NMR parameters, the first evidence of a possible relationship between the size of the crystals and T1 was demonstrated. The effects of a liquid phase on the fat crystal network were investigated with liquid tricaprin and solid tristearin. It was demonstrated that the two triacylglycerols can cocrystallize, and that the liquid phase modified the polymorphic behavior of tristearin. The evolution of T1 over time could be related to the Ostwald ripening phenomenon. In view of this evidence, it was concluded that there was a relationship between T1 and the size of the crystals in the fat crystal network.

J. Am. Chem. Soc., 2009, 131 (39), pp 13992–13999

Low Temperature 65Cu NMR Spectroscopy of the Cu+ Site in Azurin
Andrew S. Lipton†, Robert W. Heck†, Wibe A. de Jong†, Amy R. Gao†, Xiongjian Wu‡, Adrienne Roehrich‡, Gerard S. Harbison*‡ and Paul D. Ellis*†

Abstract
65Cu central-transition NMR spectroscopy of the blue copper protein azurin in the reduced Cu(I) state, conducted at 18.8 T and 10 K, gave a strongly second order quadrupole perturbed spectrum, which yielded a 65Cu quadrupole coupling constant of ±71.2 ± 1 MHz, corresponding to an electric field gradient of ±1.49 atomic units at the copper site, and an asymmetry parameter of approximately 0.2. Quantum chemical calculations employing second order Møller−Plesset perturbation theory and large basis sets successfully reproduced these experimental results. Sensitivity and relaxation times were quite favorable, suggesting that NMR may be a useful probe of the electronic state of copper sites in proteins.

J. Am. Chem. Soc., 2009, 131 (38), pp 13722–13727

Enhanced In situ Continuous-Flow MAS NMR for Reaction Kinetics in the Nanocages
Shutao Xu†‡, Weiping Zhang*†, Xianchun Liu†, Xiuwen Han† and Xinhe Bao*†

Abstract
A new approach of in situ continuous-flow laser-hyperpolarized 129Xe MAS NMR together with 13C MAS NMR is designed and applied successfully to study the adsorption and reaction kinetics in the nanospace. Methanol conversion in CHA nanocages has been investigated in detail for proof of principle demonstrating the prospect of in situ NMR of reaction kinetics. Our findings well elucidates that the reaction intermediate can be identified by 13C MAS NMR spectroscopy, meanwhile the kinetic and dynamic processes of methanol adsorption and reaction in CHA nanocages can be monitored by one- and two-dimensional hyperpolarized 129Xe MAS NMR spectroscopy under the continuous-flow condition close to the real heterogeneous catalysis. The kinetic curves and apparent activation energy of the nanocages involving the active site are obtained quantitatively. The advantages of hyperpolarized 129Xe with much higher sensitivity and shorter acquisition time allow the kinetics to be probed in a confined geometry under real working conditions.

J. Am. Chem. Soc., 2009, 131 (38), pp 13703–13708

Probing Surface Accessibility of Proteins Using Paramagnetic Relaxation in Solid-State NMR Spectroscopy
Rasmus Linser†, Uwe Fink† and Bernd Reif*†‡

Abstract
Paramagnetic Relaxation Enhancement (PRE) can be used to accelerate NMR data acquisition by reducing the longitudinal proton relaxation time T1 in the solid state. We show that the presence of paramagnetic compounds in the bulk solvent induces a site-specific relaxation in addition to local dynamics, which is dependent on the surface accessibility of the respective amide proton in the protein. Differentiation between paramagnetic relaxation and dynamics was achieved by a comparison of 1H T1 times obtained from microcrystalline protein samples prepared with different concentrations of the CuII(edta) chelate. We find that relaxation can in addition be mediated by hydroxyl groups, which transfer relaxation by their ability to exchange with the quickly relaxing bulk solvent. Furthermore, relaxation seems to be transferred by water molecules which diffuse into the protein structure and yield an efficient difference PRE in flexible regions of the protein. The experiments are demonstrated using a perdeuterated sample of the α-spectrin SH3 domain, which was microcrystallized from a buffer containing 90% D2O. Deuteration is a prerequisite to avoid spin diffusion which would otherwise compromise site specific resolution.

J. Am. Chem. Soc., 2009, 131 (38), pp 13690–13702

Dynamics of Reassembled Thioredoxin Studied by Magic Angle Spinning NMR: Snapshots from Different Time Scales
Jun Yang†§, Maria Luisa Tasayco‡ and Tatyana Polenova†

Abstract
Solid-state NMR spectroscopy can be used to probe internal protein dynamics in the absence of the overall molecular tumbling. In this study, we report 15N backbone dynamics in differentially enriched 1−73(U-13C,15N)/74−108(U-15N) reassembled thioredoxin on multiple time scales using a series of 2D and 3D MAS NMR experiments probing the backbone amide 15N longitudinal relaxation, 1H−15N dipolar order parameters, 15N chemical shift anisotropy (CSA), and signal intensities in the temperature-dependent and 1H T2′-filtered NCA experiments. The spin-lattice relaxation rates R1 (R1 = 1/T1) were observed in the range from 0.012 to 0.64 s−1, indicating large site-to-site variations in dynamics on pico- to nanosecond time scales. The 1H−15N dipolar order parameters, S, and 15N CSA anisotropies, δσ, reveal the backbone mobilities in reassembled thioredoxin, as reflected in the average S = 0.89 ± 0.06 and δσ = 92.3 ± 5.2 ppm, respectively. From the aggregate of experimental data from different dynamics methods, some degree of correlation between the motions on the different time scales has been suggested. Analysis of the dynamics parameters derived from these solid-state NMR experiments indicates higher mobilities for the residues constituting irregular secondary structure elements than for those located in the α-helices and β-sheets, with no apparent systematic differences in dynamics between the α-helical and β-sheet residues. Remarkably, the dipolar order parameters derived from the solid-state NMR measurements and the corresponding solution NMR generalized order parameters display similar qualitative trends as a function of the residue number. The comparison of the solid-state dynamics parameters to the crystallographic B-factors has identified the contribution of static disorder to the B-factors. The combination of longitudinal relaxation, dipolar order parameter, and CSA line shape analyses employed in this study provides snapshots of dynamics and a new insight on the correlation of these motions on multiple time scales.

J. Am. Chem. Soc., 2009, 131 (38), pp 13651–13658

Probing the Dynamics of a Protein Hydrophobic Core by Deuteron Solid-State Nuclear Magnetic Resonance Spectroscopy
Liliya Vugmeyster*†, Dmitry Ostrovsky†, Joseph J. Ford‡, Sarah D. Burton‡, Andrew S. Lipton‡, Gina L. Hoatson§ and Robert L. Vold§

Abstract
With the goal of investigating dynamical features of hydrophobic cores of proteins over a wide range of temperatures, the chicken villin headpiece subdomain protein (HP36) was labeled at a “single” site corresponding to any one of the two CδD3 groups of leucine-69, which is located in a key position of the core. The main techniques employed are deuteron NMR quadrupolar echo line shape analysis, and T1Z (Zeeman) and T1Q (quadrupolar order) relaxation experiments performed at 11.7 and 17.6 T over the temperature range of 112 to 298 K. The experimental data are compared with computer simulations. The deuteron line shapes give an excellent fit to a three-mode motional model that consists of (a) fast three-site rotational jumps about the pseudo C3 methyl spinning axis, (b) slower reorientation of the spinning axis, described by diffusion along a restricted arc, and (c) large angle jumps between traces of rotameric conformers. Relaxation behavior is described by a phenomenological distribution of activation energies for three-site hops at high temperatures that collapses to a single, distinctly smaller value for lower temperatures.

Chem. Mater., 2009, 21 (18), pp 4127–4134

Insights into Oxygen Exchange Between Gaseous O2 and Supported Vanadium Oxide Catalysts via 17O NMR
Christopher A. Klug*†‡, Scott Kroeker§, Pedro M. Aguiar§, Min Zhou†, Donald F. Stec† and Israel E. Wachs#

Abstract
Vanadium oxide reference compounds, KVO3 and V2O5, and supported vanadium oxide catalysts (Al2O3, TiO2, and SiO2) were investigated using magic angle sample spinning 17O NMR. All samples were 17O-enriched using gas−solid exchange. Extraction of chemical shift and quadrupolar coupling information for the model compounds KVO3 and V2O5 was performed via the simulation of MAS spectra obtained in one-pulse experiments and the observations were consistent with their known bulk structures. For the supported vanadia catalysts, it was found that the oxygen exchange process is dominated by 17O signal from the catalyst oxide supports. Spectra obtained via rotor-synchronized spin echoes revealed additional wide lines for Al2O3 and TiO2 supported vanadia catalysts that arise from 17O in the surface vanadia species of the catalysts. Additional 17O−51V TRAPDOR (TRAnsfer of Populations in DOuble Resonance) experiments support this assignment. The wide lines suggest that the local environments of the 17O nuclei associated with the dehydrated surface vanadia species are extremely heterogeneous and fall in the range of oxygen in singly (V═O) and/or doubly coordinated environments (V−O−V or V−O−Support). The relatively small total amount of 17O associated with the surface vanadia species contrasts with oxygen exchange models which commonly assume only the surface vanadium oxide layer is involved. These results demonstrate that the isotopic exchange of molecular O2 with supported metal oxide catalysts, especially supported vanadia catalysts, is a much more complex process than originally perceived.

J. Am. Chem. Soc., 2009, 131 (37), pp 13508–13515

Crystal Structure of Ammonia Monohydrate Phase II
A. Dominic Fortes*†‡, Emmanuelle Suard§, Marie-Hlne Leme-Cailleau§, Christopher J. Pickard and Richard J. Needs

Abstract
We have determined the crystal structure of ammonia monohydrate phase II (AMH II) employing a combination of ab initio computational structure prediction and structure solution from neutron powder diffraction data using direct space methods. Neutron powder diffraction data were collected from perdeuterated AMH II using the D2B high-resolution diffractometer at the Institut Laue-Langevin. AMH II crystallizes in space-group Pbca with 16 formula units in a unit-cell of dimensions a = 18.8285(4) Å, b = 6.9415(2) Å, c = 6.8449(2) Å, and V = 894.61(3) Å3 [ρcalcdeuterated = 1187.56(4) kg m−3] at 502 MPa, 180 K. The structure is characterized by sheets of tessellated pentagons formed by orientationally ordered O−D···O, O−D···N, and N−D···O hydrogen-bonds; these sheets are stacked along the a-axis and connected by N−D···O hydrogen bonds alone. With the exception of the simple body-centered-cubic high-pressure phases of ammonia monohydrate and ammonia dihydrate, this is the first complex molecular structure of any of the high-pressure stoichiometric ammonia hydrates to be determined. The powder structure solution is complemented by an ab initio structure prediction using density functional theory which gives an almost identical hydrogen bonding network.

J. Am. Chem. Soc., 2009, 131 (37), pp 13430–13440

Implementation of High Resolution 43Ca Solid State NMR Spectroscopy: Toward the Elucidation of Calcium Sites in Biological Materials
Danielle Laurencin*†‡, Christel Gervais§, Alan Wong†, Cristina Coelho§, Francesco Mauri, Dominique Massiot#, Mark E. Smith† and Christian Bonhomme*§

Abstract
Calcium is one of the most abundant cations in living organisms. It is found in the mineral phase of bone and in proteins like calmodulin. However, its exact environment beyond the first coordination sphere is often unknown, thus hampering the understanding of many biological processes. Here, calcium benzoate trihydrate (Ca(C6H5COO)2·3H2O) was used as a model for the NMR analysis of calcium sites in biological materials, because of the similarity of its calcium coordination, to water and carboxylate ligands, to that in several calcium-proteins. First, calcium-43 magic angle spinning (MAS) and static NMR spectra of a 43Ca enriched sample were recorded at different magnetic fields, to investigate the electronic environment of calcium. Complex static lineshapes were obtained because of the presence of anisotropic NMR interactions of similar magnitude (chemical shift anisotropy and quadrupolar interaction), and the full interpretation of the spectra required simulations and gauge-including projector augmented wave (GIPAW) DFT calculations. An NMR investigation of the coordination environment of Ca2+ was carried out, using high resolution 13C−43Ca MAS NMR experiments such as TRAPDOR (transfer of population double resonance) and heteronuclear J-spin−echoes. It was shown that despite the weakness of 13C−43Ca interactions, it is possible to discriminate carbon atoms according to their calcium environment. Long-range calcium−carbon correlations were even evidenced by TRAPDOR, reaching distances >5.6 Å. This work demonstrates that by combining solid state NMR experiments, DFT calculations, and simulations, it will be possible to elucidate the electronic and coordination environment of calcium in many important and complex materials.

J. Am. Chem. Soc., 2009, 131 (37), pp 13228–13229

13C−13C Correlation Spectroscopy of Membrane-Associated Influenza Virus Fusion Peptide Strongly Supports a Helix-Turn-Helix Motif and Two Turn Conformations

Yan Sun and David P. Weliky*

Abstract
The influenza virus fusion peptide (IFP) is the N-terminal domain of the viral hemagglutinin protein, binds to the endosomal membrane, and plays a critical role in fusion between the viral and endosomal membranes which is a primary step in infection. The IFP is also an important system for testing simulation methods for membrane-associated peptides. In detergent, the IFP forms helix-turn-helix and helix-turn-strand structures at pH 5.0 and 7.4, respectively, while simulations in membranes by different groups have yielded conflicting results with some reports of a continuous helix without a turn. In this study, 13C−13C NMR correlation spectra were obtained for the membrane-associated IFP and the 13C chemical shifts supported a helix-turn-helix motif at both pH 5.0 and 7.4 with an alternate turn conformation at pH 5.0 that was absent at pH 7.4. The alternate conformation was correlated with protonation of the side chain of Glu-11 in the turn and with greater fusion at pH 5.0. The structures are overall consistent with the hypothesis of “inverted V” membrane location of the IFP with insertion of the N-terminal region into the membrane and contact of the turn with the lipid/water interface. The positions of hydrophobic residues in the pH 5.0 structure may favor membrane insertion with resultant increased membrane perturbation and fusion rate. In addition to their functional relevance, these IFP structures are important reference data for simulations of the membrane-associated IFP which can in principle detect the full conformational distribution of the IFP.

Thursday, October 15, 2009

Cryst. Growth Des., 2009, 9 (2), pp 921–937

Solid-State NMR Analysis of Organic Cocrystals and Complexes
Frederick G. Vogt*†, Jacalyn S. Clawson†, Mark Strohmeier†, Andrew J. Edwards‡, Tran N. Pham‡ and Simon A. Watson‡

Abstract
Solid-state NMR (SSNMR) is capable of providing detailed structural information about organic and pharmaceutical cocrystals and complexes. SSNMR nondestructively analyzes small amounts of powdered material and generally yields data with higher information content than vibrational spectroscopy and powder X-ray diffraction methods. These advantages can be utilized in the analysis of pharmaceutical cocrystals, which are often initially produced using solvent drop grinding techniques that do not lend themselves to single crystal growth for X-ray diffraction studies. In this work, several molecular complexes and cocrystals are examined to understand the capabilities of the SSNMR techniques, particularly their ability to prove or disprove molecular association and observe structural features such as hydrogen bonding. Dipolar correlation experiments between spin pairs such as 1H−1H, 1H−13C, and 19F−13C are applied to study hydrogen bonding, intermolecular contacts, and spin diffusion to link individual molecules together in a crystal structure and quickly prove molecular association. Analysis of the principal components of chemical shift tensors is also utilized where relevant, as these are more sensitive to structural effects than the isotropic chemical shift alone. In addition, 1H T1 relaxation measurements are also demonstrated as a means to prove phase separation of components. On the basis of these results, a general experimental approach to cocrystal analysis by SSNMR is suggested.

J. Am. Chem. Soc., 2009, 131 (33), pp 11939–11948

Measurement of Methyl Axis Orientations in Invisible, Excited States of Proteins by Relaxation Dispersion NMR Spectroscopy
Andrew J. Baldwin, D. Flemming Hansen, Pramodh Vallurupalli and Lewis E. Kay*

Abstract
Few detailed studies of transiently populated conformations of biological molecules have emerged despite the fact that such states are often important to processes such as protein folding, enzyme catalysis, molecular recognition and binding. A major limitation has been the lack of experimental tools to study these often invisible, short-lived conformers. Recent advances in relaxation dispersion NMR spectroscopy are changing this paradigm with the potential to generate high resolution structural information which is necessary for a rigorous characterization of these states. In this study, we present an experimental method for establishing the relative orientations of methyl groups in invisible, excited states of proteins by measuring methyl 1H−13C residual dipolar couplings (RDCs). In our approach, four two-dimensional spectra are acquired at a pair of static magnetic fields. Each spectrum contains one of the four isolated multiplet components of a coupled methyl carbon, whose signal intensities, modulated by the pulsing frequency of a Carr−Purcell−Meiboom−Gill (CPMG) element, are sensitive to both chemical shift and RDC differences between exchanging states. In addition, data sets from a CPMG experiment which monitors the decay of in-phase methyl 13C magnetization are recorded, that are sensitive only to the differences in chemical shifts between the states. Using our methodology, RDC values obtained from an invisible state in an exchanging system are shown to be in good agreement with the corresponding values measured under conditions where the invisible state is stabilized to become the highly populated ground state. The approach allows the measurement of anisotropic restraints at methyl positions in excited states and complements previously developed experiments focusing on the protein backbone.

J. Am. Chem. Soc., 2009, 131 (33), pp 11861–11874

31P MAS Refocused INADEQUATE Spin−Echo (REINE) NMR Spectroscopy: Revealing J Coupling and Chemical Shift Two-Dimensional Correlations in Disordered Solids
Paul Guerry†, Mark E. Smith and Steven P. Brown*

Abstract
Two-dimensional (2D) variations in 2JP1,P1, 2JP1,P2, and 2JP2,P2 are obtained—using the REINE (REfocused INADEQUATE spin−Echo) pulse sequence presented by Cadars et al. (Phys. Chem. Chem. Phys. 2007, 9, 92−103)—from pixel-by-pixel fittings of the spin−echo modulation for the 2D correlation peaks due to linked phosphate tetrahedra (P1−P1, P1−P2, P2−P1, and P2−P2) in a 31P refocused INADEQUATE solid-state MAS NMR spectrum of a cadmium phosphate glass, 0.575CdO−0.425P2O5. In particular, separate variations for each 2D 31P REINE peak are obtained which reveal correlations between the J couplings and the 31P chemical shifts of the coupled nuclei that are much clearer than those evident in previously presented 2D z-filtered 31P spin−echo spectra. Notably, such correlations between the J couplings and the 31P chemical shifts are observed even though the conditional probability distributions extracted using the protocol of Cadars et al. (J. Am. Chem. Soc. 2005, 127, 4466−4476) indicate that there is no marked correlation between the 31P chemical shifts of neighboring phosphate tetrahedra. For 2D peaks at the P2 31P chemical shift in the direct dimension, there can be contributions from chains of three units (P1−P2−P1), chains of four units (P1−P2−P2−P1), or longer chains or rings (−P2−P2−P2−): for the representative glass considered here, best fits are obtained assuming a glass comprised predominantly of chains of four units. The following variations are found: 2JP1,P1 = 13.4 ± 0.3 to 14.8 ± 0.5 Hz, 2JP1,P2 = 15.0 ± 0.3 to 18.2 ± 0.3 Hz, and 2JP2,P2 = 5.9 ± 0.6 to 9.1 ± 0.9 Hz from the fits to the P1−P1, P1−P2, and P2−P2 peaks, respectively. The correlation of a particular J coupling with the 31P chemical shifts of the considered nucleus and the coupled nucleus is quantified by the coefficients CF2 and CF1 that correspond to the average pixel-by-pixel change in the J coupling with respect to the chemical shift of the observed (F2) and neighboring (F1) 31P nuclei, respectively.

J. Am. Chem. Soc., 2009, 131 (33), pp 11855–11860

Direct Interaction between Amphotericin B and Ergosterol in Lipid Bilayers As Revealed by 2H NMR Spectroscopy
Nobuaki Matsumori, Kazuaki Tahara, Hiroko Yamamoto, Atsushi Morooka, Mototsugu Doi, Tohru Oishi and Michio Murata

Abstract
Although amphotericin B (AmB) is thought to exert its antifungal activity by forming transmembrane ion-permeable self-assemblies together with ergosterol, no previous study has directly proven AmB−ergosterol interaction. To establish the interaction, we measured 2H NMR using deuterium-labeled sterols and AmB. The 2H NMR spectra of deuterated ergosterol in palmitoyloleoylphosphatidylcholine (POPC) bilayers showed that fast axial diffusion of erogosterol was almost completely inhibited by the coexistence of AmB. Conversely, cholesterol mobility in POPC membrane was essentially unchanged with or without AmB. These results unequivocally demonstrate that ergosterol has significant interaction with AmB in POPC bilayers. In addition, we examined the mobility of AmB using deuterium-labeled AmB, and found that, although AmB is almost immobilized in sterol-free and cholesterol-containing POPC membranes, a certain ratio of AmB molecules acquires mobility in the presence of ergosterol. The similar mobility of AmB and ergosterol in POPC bilayers confirmed the idea of the direct intermolecular interaction between ergosterol and AmB.

J. Am. Chem. Soc., 2009, 131 (33), pp 11801–11810

Solid-State NMR Investigations of the Immobilization of a BF4− Salt of a Palladium(II) Complex on Silica
Jerzy W. Wiench, Christophe Michon, Arkady Ellern, Paul Hazendonk, Adriana Iuga, Robert J. Angelici and Marek Pruski

Abstract
The structure of the silica supported palladium(II) complex [Pd(dppp)(S2C-NEt2)]BF4 (abbreviated as [Pd(dppp)(dtc)]BF4, where dppp is Ph2P(CH2)3PPh2) and interactions between the [Pd(dppp)(dtc)]+ cation, the BF4− anion, and the silica surface are studied using solid-state NMR spectroscopy. The unsupported, crystalline form of [Pd(dppp)(dtc)]BF4 is also investigated, both by X-ray diffraction and NMR. The structures of the cation and anion are found to be essentially the same in both unsupported and supported complex. The [Pd(dppp)(dtc)]BF4 loading has been determined by quantitative measurements of 11B, 19F, and 31P intensities, whereas the arrangement of anions and cations on the surface of silica has been established by two-dimensional heteronuclear correlation experiments involving 1H, 11B, 13C, 19F, 29Si, and 31P nuclei. At low coverages, the [Pd(dppp)(dtc)]+ cations are located near the BF4− anions, which in turn are immobilized directly on the surface near the Q4 sites. At higher loadings, which in this study corresponded to 0.06−0.15 mmol/g, the complexes stack on top of each other, despite the fact that the directly adsorbed molecules take up less than 10% of the silica surface. The relevance of these findings to heterogeneous catalysis is discussed.

J. Am. Chem. Soc., 2009, 131 (33), pp 11762–11769

89Y and 13C NMR Cluster and Carbon Cage Studies of an Yttrium Metallofullerene Family, Y3N@C2n (n = 40−43)
Wujun Fu, Liaosa Xu, Hugo Azurmendi, Jiechao Ge, Tim Fuhrer, Tianming Zuo, Jonathan Reid, Chunying Shu, Kim Harich and Harry C. Dorn*

Abstract
The members of a new family of yttrium trimetallic nitride-templated (TNT) endohedral metallofullerenes (EMFs), Y3N@C2n (n = 40−43), have been synthesized and purified. On the basis of experimental and computational 13C NMR studies, we propose cage structures for Y3N@Ih-C80 (IPR allowed), Y3N@D5h-C80 (IPR allowed), Y3N@Cs-C82 (non-IPR), Y3N@Cs-C84 (non-IPR), and Y3N@D3-C86 (IPR allowed). A significant result is the limited number of isomers found for each carbon cage. For example, there are 24 isolated pentagon rule (IPR) and 51568 non-IPR structures possible for the C84 cage, but only one major isomer of Y3N@Cs-C84 was found. The current study confirms the unique role of the trimetallic nitride (M3N)6+ cluster template in the Krtschmer−Huffman electric-arc process for fullerene cage size and high symmetry isomer selectivity. This study reports the first 89Y NMR results for Y3N@Ih-C80, Y3N@Cs(51365)-C84, and Y3N@D3(19)-C86, which reveal a progression from isotropic to restricted (Y3N)6+ cluster motional processes. Even more surprising is the sensitivity of the 89Y NMR chemical shift parameter to subtle changes in the electronic environment at each yttrium nuclide in the (Y3N)6+ cluster (more than 200 ppm for these EMFs). This 89Y NMR study suggests that 89Y NMR will evolve as a powerful tool for cluster motional studies of EMFs.

Tuesday, October 13, 2009

J. Am. Chem. Soc., 2009, 131 (35), pp 12745–12754

Measurement of Methyl Group Motional Parameters of Invisible, Excited Protein States by NMR Spectroscopy
D. Flemming Hansen, Pramodh Vallurupalli and Lewis E. Kay*

Abstract
An understanding of many biological processes can only be achieved through studies of the structure (enthalpy) and motions (entropy) of the key molecules that are involved, including those that are formed only transiently and with low population. These transiently formed, low populated states are invisible to most biophysical techniques but in many cases they can be studied in detail using relaxation dispersion NMR spectroscopy. Relaxation dispersion methodology has recently been described for the measurement of protein backbone excited state chemical shifts as well as bond vector orientations, which form the basis for structural studies of these invisible conformers. It is of interest to extend such studies by quantifying motional parameters of the excited state, providing a more complete description of the energy landscape that drives the biochemical event in question. Herein we describe a relaxation dispersion method for measuring site-specific motional parameters of methyl containing residues in the excited state. The approach is applied to the invisible unfolded state of the G48M Fyn SH3 domain that is in exchange with the folded conformation. Not surprisingly, the degree of disorder is in general higher in the unfolded state than in the folded conformer, although there is some ordering of side-chains in the unfolded state toward the C-terminal region of the domain. The development of the present methodology provides the first step toward characterizing the motional properties of invisible conformers, complementing the structural information that is already available from relaxation dispersion studies.

Cryst. Growth Des., 2009, 9 (9), pp 4051–4059

Solid-State NMR and X-ray Diffraction Study of Structure and Dynamics of Dihydrate and Anhydrous Form of Tyr-Ala-Phe
Katarzyna Trzeciak-Karlikowska, Anna Bujacz, Agata Jeziorna, Włodzimierz Ciesielski†, Grzegorz D. Bujacz, Jarosław Gajda†, Danuta Pentak and Marek J. Potrzebowski*

Tyr-d-Ala-Phe is a “message sequence” of naturally occurring opioid peptides, deltorphin I (Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH2), deltorphin II (Tyr-d-Ala-Phe-Glu-Val-Val-Gly-NH2), and dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2). Analogous heptapeptides containing l-alanine instead of d-alanine are not biologically active. In a previous paper (J. Phys. Chem. B 2004, 108 (14), 4535−4545), we reported X-ray and NMR data for Tyr-d-Ala-Phe. In the current report, we present structural studies of Tyr-Ala-Phe, a “false message sequence” of opioid peptides. It has been found that Tyr-Ala-Phe crystallizes in two forms, as anhydrate (Form I) and dihydrate (Form II). Crystal and molecular structure of both forms was established by means of low-temperature X-ray measurements. Form I is orthorhombic with space group P212121, while II is hexagonal with space group P65. Solid-state NMR was employed to study the structure and molecular dynamics of I and II. Analysis of cross-polarization buildup curves and 13C chemical shift tensor (CST) parameters obtained by a two-dimensional PASS experiment have revealed a dramatic difference in the molecular motion of both modifications. 13C T1 relaxation times have provided further evidence confirming distinct molecular dynamics. The attempt to understand the role of the stereochemistry of Ala residue in opioid peptide sequences in relation to intramolecular interactions and preorganization mechanisms is presented.

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