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-dimensional nuclear magnetic resonance (2D NMR) spectroscopy provides the means to extract diverse physical, chemical, and biological information at an atomic level. Conventional sampling schemes, however, may result in relatively long 2D experiments; this has stimulated the search for alternative, rapid acquisition schemes. Among the strategies that have been recently proposed for achieving this counts an “ultrafast” approach, relying on the spatial encoding of the indirect domain evolution to provide arbitrary spectra within a single scan. A common feature of all spatial encoding schemes hitherto described is their uniform encoding of a continuous bandwidth of indirect-domain frequencies, regardless of the chemical sites' spectral distribution within it. These very general conditions, however, are often associated with a number of tradeoffs and compromises in the spectral widths and resolutions that can be achieved for both the direct and indirect domains. This paper proposes a different strategy for single-scan acquisition of 2D spectra, which performs an optimal encoding by employing a priori information regarding the positions of NMR resonances along the indirect domain. We denote this as “spatial/spectral encoding”; the underlying principles of this new approach, together with experimental results based on uni- and multidimensional 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.

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.

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.

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.