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.

J. Am. Chem. Soc., Article ASAP

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.

J. Am. Chem. Soc., Article ASAP

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., 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

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.

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.

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.