Publications by authors named "Wolfgang Bermel"

91 Publications

Faster and cleaner real-time pure shift NMR experiments.

J Magn Reson 2015 Oct 31;259:207-15. Epub 2015 Aug 31.

Institute of Chemistry/Organic and Bioorganic Chemistry, University of Graz, Austria. Electronic address:

Real-time pure shift experiments provide highly resolved proton NMR spectra which do not require any special processing. Although being more sensitive than their pseudo 2D counterparts, their signal intensities per unit time are still far below regular NMR spectra. In addition, scalar coupling evolution during the individual data chunks produces decoupling sidebands. Here we show that faster and cleaner real-time pure shift spectra can be obtained through the implementation of two parameter alterations. Variation of the FID chunk lengths between individual transients significantly suppresses decoupling sidebands for any kind of real-time pure shift spectra and thus allows for example the analysis of minor components in compound mixtures. Shifting the excitation frequency between individual scans of real-time slice-selective pure shift spectra increases their sensitivity obtainable in unit time by allowing faster repetitions of acquisitions.
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http://dx.doi.org/10.1016/j.jmr.2015.08.011DOI Listing
October 2015

A different approach to multiplicity-edited heteronuclear single quantum correlation spectroscopy.

J Magn Reson 2015 Oct 8;259:82-6. Epub 2015 Aug 8.

Bruker BioSpin GmbH, Silberstreifen, D-76287 Rheinstetten, Germany. Electronic address:

A new experiment for recording multiplicity-edited HSQC spectra is presented. In standard multiplicity-edited HSQC experiments, the amplitude of CH2 signals is negative compared to those of CH and CH3 groups. We propose to reverse the sign of (13)C frequencies of CH2 groups in t1 as criteria for editing. Basically, a modified [BIRD](r,x) element (Bilinear Rotation Pulses and Delays) is inserted in a standard HSQC pulse sequence with States-TPPI frequency detection in t1 for this purpose. The modified BIRD element was designed in such a way as to pass or stop the evolution of the heteronuclear (1)JHC coupling. This is achieved by adding a 180° proton RF pulse in each of the 1/2J periods. Depending on their position the evolution is switched on or off. Usually, the BIRD- element is applied on real and imaginary increments of a HSQC experiment to achieve the editing between multiplicities. Here, we restrict the application of the modified BIRD element to either real or imaginary increments of the HSQC. With this new scheme for editing, changing the frequency and/or amplitude of the CH2 signals becomes available. Reversing the chemical shift axis for CH2 signals simplifies overcrowded frequency regions and thus avoids accidental signal cancellation in conventional edited HSQC experiments. The practical implementation is demonstrated on the protein Lysozyme. Advantages and limitations of the idea are discussed.
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http://dx.doi.org/10.1016/j.jmr.2015.07.006DOI Listing
October 2015

Homodecoupled 1,1- and 1,n-ADEQUATE: Pivotal NMR Experiments for the Structure Revision of Cryptospirolepine.

Angew Chem Int Ed Engl 2015 Aug 15;54(35):10160-4. Epub 2015 Jul 15.

Process & Analytical Chemistry, Merck Research Laboratories, Mailstop: 800-D133, 126 E. Scott Ave, Rahway, NJ 07065 (USA).

Cryptospirolepine is the most structurally complex alkaloid discovered and characterized thus far from any Cryptolepis specie. Characterization of several degradants of the original, sealed NMR sample a decade after the initial report called the validity of the originally proposed structure in question. We now report the development of improved, homodecoupled variants of the 1,1- and 1,n-ADEQUATE (HD-ADEQUATE) NMR experiments; utilization of these techniques was critical to successfully resolving long-standing structural questions associated with crytospirolepine.
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http://dx.doi.org/10.1002/anie.201502540DOI Listing
August 2015

Cd NMR Experiments Reveal an Unusual Metal Cluster in the Solution Structure of the Yeast Splicing Protein Bud31p.

Angew Chem Weinheim Bergstr Ger 2015 Apr 20;127(16):4943-4946. Epub 2015 Feb 20.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH (UK).

Establishing the binding topology of structural zinc ions in proteins is an essential part of their structure determination by NMR spectroscopy. Using Cd NMR experiments with Cd-substituted samples is a useful approach but has previously been limited mainly to very small protein domains. Here we used Cd NMR spectroscopy during structure determination of Bud31p, a 157-residue yeast protein containing an unusual ZnCys cluster, demonstrating that recent hardware developments make this approach feasible for significantly larger systems.
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http://dx.doi.org/10.1002/ange.201412210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4954022PMC
April 2015

Engineering of an all-heteronuclear 5-qubit NMR quantum computer.

Magn Reson Chem 2015 Jun 9;53(6):442-7. Epub 2015 Apr 9.

Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747, Garching, Germany.

The realization of an all-heteronuclear 5-qubit nuclear magnetic resonance quantum computer is reported, from the design and synthesis of a suitable molecule through the engineering of a prototype 6-channel probe head. Full control over the quantum computer is shown by a benchmark experiment.
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http://dx.doi.org/10.1002/mrc.4233DOI Listing
June 2015

¹¹³Cd NMR experiments reveal an unusual metal cluster in the solution structure of the yeast splicing protein Bud31p.

Angew Chem Int Ed Engl 2015 Apr 20;54(16):4861-4. Epub 2015 Feb 20.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH (UK).

Establishing the binding topology of structural zinc ions in proteins is an essential part of their structure determination by NMR spectroscopy. Using (113)Cd NMR experiments with (113)Cd-substituted samples is a useful approach but has previously been limited mainly to very small protein domains. Here we used (113)Cd NMR spectroscopy during structure determination of Bud31p, a 157-residue yeast protein containing an unusual Zn3Cys9 cluster, demonstrating that recent hardware developments make this approach feasible for significantly larger systems.
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http://dx.doi.org/10.1002/anie.201412210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471582PMC
April 2015

"CON-CON" assignment strategy for highly flexible intrinsically disordered proteins.

J Biomol NMR 2014 Dec 19;60(4):209-18. Epub 2014 Oct 19.

CERM and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy.

Intrinsically disordered proteins (IDPs) are a class of highly flexible proteins whose characterization by NMR spectroscopy is complicated by severe spectral overlaps. The development of experiments designed to facilitate the sequence-specific assignment procedure is thus very important to improve the tools for the characterization of IDPs and thus to be able to focus on IDPs of increasing size and complexity. Here, we present and describe the implementation of a set of novel ¹H-detected 5D experiments, (HACA)CON(CACO)NCO(CA)HA, BT-(H)NCO(CAN)CONNH and BT-HN(COCAN)CONNH, optimized for the study of highly flexible IDPs that exploit the best resolved correlations, those involving the carbonyl and nitrogen nuclei of neighboring amino acids, to achieve sequence-specific resonance assignment. Together with the analogous recently proposed pulse schemes based on ¹³C detection, they form a complete set of experiments for sequence-specific assignment of highly flexible IDPs. Depending on the particular sample conditions (concentration, lifetime, pH, temperature, etc.), these experiments present certain advantages and disadvantages that will be discussed. Needless to say, that the availability of a variety of complementary experiments will be important for accurate determination of resonance frequencies in complex IDPs.
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http://dx.doi.org/10.1007/s10858-014-9867-6DOI Listing
December 2014

HCNMBC--a pulse sequence for H-(C)-N Multiple Bond Correlations at natural isotopic abundance.

J Magn Reson 2014 Oct 12;247:38-41. Epub 2014 Aug 12.

Bruker UK Limited, Banner Lane, Coventry CV4 9GH, UK. Electronic address:

We propose a pulse sequence, HCNMBC for multiple-bond H-(C)-N correlation experiments via one-bond (1)J(C,H) and one- or multiple bond (n)J(N,C) coupling constants (typically n=1-3) at the natural isotopic abundance. A new adiabatic refocussing sequence is introduced to provide accurate and robust refocussing of both chemical shift and J-evolution over wide ranges of C-13 and N-15 frequencies. It is demonstrated that the proposed pulse sequence provides high quality spectra even for sub-milligram samples. We show that when a 1.7 mm cryoprobe is available as little as 10 μg of glycine in D2O is sufficient to obtain the HCNMBC spectrum in ca. 12 h. The preliminary results indicate that the pulse sequence has a great potential in the structure determination of nitrogen heterocycles especially in cases where synthesis produces regioisomers.
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http://dx.doi.org/10.1016/j.jmr.2014.07.011DOI Listing
October 2014

Two-dimensional NMR spectroscopy with temperature-sweep.

Chemphyschem 2014 Aug 26;15(11):2217-20. Epub 2014 May 26.

Bruker BioSpin GmbH, Silberstreifen, 76287 Rheinstetten (Germany).

Two-dimensional nuclear magnetic resonance (NMR) spectroscopy is useful for studying temperature-dependent effects on molecular structure. However, experimental time is usually long, because sampling is repeated at several temperatures. A novel solution to the problem is proposed, in which signal sampling is performed in parallel to the linear temperature-sweep.
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http://dx.doi.org/10.1002/cphc.201402191DOI Listing
August 2014

Improving the sensitivity of conventional spin echo spectra by preservation of initial signal-to-noise ratio.

J Magn Reson 2014 May 7;242:220-3. Epub 2014 Mar 7.

Bruker BioSpin GmbH, Silberstreifen, D-76287 Rheinstetten, Germany. Electronic address:

A simple processing strategy is introduced to enhance the spectral quality and signal-to-noise ratio in conventional J resolved spectra. The idea of pseudo echo filtering is extended to conserve the primary signal-to-noise, predominating at the beginning of the FID in the indirect dimension. This is achieved by matching the maximum amplitude of the FID with that of the sine window function. Practically, the FID is right shifted by the number of acquired points in the indirect dimension; missing data points are backward predicted and finally multiplied with the unshifted sine window function. Standard processing tools are employed for this purpose. The results of data processing using different window functions with and without right shifts and back predictions are discussed. The signal-to-noise ratio of the J resolved spectrum is increased by a factor of 6 compared to standard data processing using pseudo echo filtering alone.
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http://dx.doi.org/10.1016/j.jmr.2014.02.024DOI Listing
May 2014

High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins.

J Biomol NMR 2013 Dec 8;57(4):353-61. Epub 2013 Nov 8.

Bruker BioSpin GmbH, Silberstreifen, 76287, Rheinstetten, Germany.

We present three novel exclusively heteronuclear 5D (13)C direct-detected NMR experiments, namely (H(N-flip)N)CONCACON, (HCA)CONCACON and (H)CACON(CA)CON, designed for easy sequence-specific resonance assignment of intrinsically disordered proteins (IDPs). The experiments proposed have been optimized to overcome the drawbacks which may dramatically complicate the characterization of IDPs by NMR, namely the small dispersion of chemical shifts and the fast exchange of the amide protons with the solvent. A fast and reliable automatic assignment of α-synuclein chemical shifts was obtained with the Tool for SMFT-based Assignment of Resonances (TSAR) program based on the information provided by these experiments.
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http://dx.doi.org/10.1007/s10858-013-9793-zDOI Listing
December 2013

Broadband inversion of 1J(CC) responses in 1,n-ADEQUATE spectra.

J Magn Reson 2013 Nov 9;236:126-33. Epub 2013 Aug 9.

Merck Research Laboratories, Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structure Elucidation Group, Rahway, NJ 07065, USA. Electronic address:

Establishing the carbon skeleton of a molecule greatly facilitates the process of structure elucidation, both manual and computer-assisted. Recent advances in the family of ADEQUATE experiments demonstrated their potential in this regard. 1,1-ADEQUATE, which provides direct (13)C-(13)C correlation via (1)J(CC), and 1,n-ADEQUATE, which typically yields (3)J(CC) and (1)J(CC) correlations, are more sensitive and more widely applicable experiments than INADEQUATE and PANACEA. A recently reported modified pulse sequence that semi-selectively inverts (1)J(CC) correlations in 1,n-ADEQUATE spectra provided a significant improvement, allowing (1)J(CC) and (n)J(CC) correlations to be discerned in the same spectrum. However, the reported experiment requires a careful matching of the amplitude transfer function with (1)J(CC) coupling constants in order to achieve the inversion, and even then some (1)J(CC) correlations could still have positive intensity due to the oscillatory nature of the transfer function. Both shortcomings limit the practicality of the method. We now report a new, dual-optimized inverted (1)J(CC) 1,n-ADEQUATE experiment, which provides more uniform inversion of (1)J(CC) correlations across the range of 29-82 Hz. Unlike the original method, the dual optimization experiment does not require fine-tuning for the molecule's (1)J(CC) coupling constant values. Even more usefully, the dual-optimized version provides up to two-fold improvement in signal-to-noise for some long-range correlations. Using modern, cryogenically-cooled probes, the experiment can be successfully applied to samples of ~1 mg under favorable circumstances. The improvements afforded by dual optimization inverted (1)J(CC) 1,n-ADEQUATE experiment make it a useful and practical tool for NMR structure elucidation and should facilitate the implementation and utilization of the experiment.
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http://dx.doi.org/10.1016/j.jmr.2013.07.016DOI Listing
November 2013

Efficient detection of hydrogen bonds in dynamic regions of RNA by sensitivity-optimized NMR pulse sequences.

Angew Chem Int Ed Engl 2013 Sep 14;52(40):10487-90. Epub 2013 Aug 14.

Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg (Germany) http://www.nmr.ch.tum.de/; Center for Integrated Protein Science Munich and Chair of Biomolecular NMR, TU München, Lichtenbergstr. 4, 85747 Garching (Germany).

Improved Sensitivity: Efficient NMR experiments are presented for determining the secondary structure in large and dynamic RNAs using J-couplings across hydrogen bonds. The experiments provide up to eight-fold improved sensitivity and thus enable detection of base pairs in dynamic regions even in large RNAs.
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http://dx.doi.org/10.1002/anie.201304391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3882076PMC
September 2013

Broadband homodecoupled NMR spectroscopy with enhanced sensitivity.

J Magn Reson 2013 Aug 30;233:92-5. Epub 2013 May 30.

NMR Laboratory of SANOFI, C&BD (Chemistry & Biotechnology Development Frankfurt Chemistry), Industriepark Hoechst, Building D770, Labor 204, D-65926 Frankfurt/Main, Germany.

A new type of broadband homodecoupling technique is described, which is based on the original version of the Zangger-Sterk experiment, but results in a spectrum with higher sensitivity. The homodecoupling is performed by a combination of selective and non-selective 180° RF pulses in the presence of weak rectangular pulsed field gradients in a pseudo 2D experiment. The proposed experiment uses a fast pulsing approach to increase the signal-to-noise ratio per unit time. The recycle delay is significantly shortened typically to about 100 ms. After each scan, the offset of the selective shaped pulse is changed to access fresh magnetisation from adjacent frequency/spatial regions. The physical acquisition time was limited to 40 ms to keep the total length of the pulse sequence as short as possible. Broadband inversion BIP pulses are used instead of 180° hard pulses. They are used pairwise to cancel out unwanted phase shifts over the bandwidth. Reconstruction of the homodecoupled spectrum was done by concatenating the first 10 ms of the FID from each single increment to obtain the final homodecoupled proton FID followed by Fourier transformation. The new method can either be used to acquire broadband homodecoupled spectra in a shorter time or to increase the signal-to-noise ratio compared to the original Zangger-Sterk experiment. Using eight different frequencies can thus lead to a signal to noise gain of a factor √8 or a factor of eight in time.
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http://dx.doi.org/10.1016/j.jmr.2013.05.008DOI Listing
August 2013

Monitoring fast reactions by spatially-selective and frequency-shifted continuous NMR spectroscopy: application to rapid-injection protein unfolding.

Chem Commun (Camb) 2013 Apr 12;49(30):3155-7. Epub 2013 Mar 12.

Institute of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria.

The repetition rate of an NMR experiment is usually limited by the longitudinal relaxation times of the investigated molecule. Here we show that continuous excitation and data acquisition, without any interscan delay, is possible by a spatially resolved experiment where different nuclei are excited in consecutive scans.
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http://dx.doi.org/10.1039/c3cc39107hDOI Listing
April 2013

Broadband homodecoupled heteronuclear multiple bond correlation spectroscopy.

J Magn Reson 2013 Mar 8;228:125-9. Epub 2013 Jan 8.

NMR Laboratory of C&BD (Chemistry & Biotechnology Development Frankfurt Chemistry), Industriepark Hoechst, Building D770, Labor 204, D-65926 Frankfurt/Main, Germany.

A general concept for removing proton-proton scalar J couplings in 2D NMR spectroscopy is proposed. The idea is based on introducing an additional J resolved dimension into the pulse sequence of a conventional 2D experiment to design a pseudo 3D NMR experiment. The practical demonstration is exemplified on the widely used gradient coherence selected heteronuclear long-range correlation spectroscopy (HMBC). We refer to this type of pulse sequence as tilt HMBC experiment. For every (13)C chemical shift evolution increment, a homonuclear J resolved experiment is recorded. The long-range defocusing delay of the HMBC pulse sequence is exploited to implement this building block. The J resolved evolution period is incremented in a way very similar to ACCORDION spectroscopy to accommodate the buildup of heteronuclear long-range antiphase magnetisation as well. After Fourier transformation in all dimensions the spectra are tilted in the J resolved dimension. Finally, a projection along the J resolved dimension is calculated leading to almost disappearance of proton-proton spin multiplicities in the 2D tilt HMBC spectrum. The tilt HMBC experiment combines sensitivity with simple experimental setup and can be recorded with short recycle delays, when combined with Ernst angle excitation. The recorded spectra display singlet proton signals for long-range correlation peaks making an unambiguous signal assignment much easier. In addition to the new experiment a simple processing technique is applied to efficiently suppress the noise originating from forward linear prediction in the indirect evolution dimensions. In case of issues with fast repetition times, probe heating and RF power handling most of the RF pulses can be replaced by broadband, frequency swept pulses operating at much lower power.
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http://dx.doi.org/10.1016/j.jmr.2012.12.018DOI Listing
March 2013

The Fantastic Four: A plug 'n' play set of optimal control pulses for enhancing NMR spectroscopy.

J Magn Reson 2013 Mar 27;228:16-31. Epub 2012 Dec 27.

Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.

We present highly robust, optimal control-based shaped pulses designed to replace all 90° and 180° hard pulses in a given pulse sequence for improved performance. Special attention was devoted to ensuring that the pulses can be simply substituted in a one-to-one fashion for the original hard pulses without any additional modification of the existing sequence. The set of four pulses for each nucleus therefore consists of 90° and 180° point-to-point (PP) and universal rotation (UR) pulses of identical duration. These 1ms pulses provide uniform performance over resonance offsets of 20kHz ((1)H) and 35kHz ((13)C) and tolerate reasonably large radio frequency (RF) inhomogeneity/miscalibration of ±15% ((1)H) and ±10% ((13)C), making them especially suitable for NMR of small-to-medium-sized molecules (for which relaxation effects during the pulse are negligible) at an accessible and widely utilized spectrometer field strength of 600MHz. The experimental performance of conventional hard-pulse sequences is shown to be greatly improved by incorporating the new pulses, each set referred to as the Fantastic Four (Fanta4).
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http://dx.doi.org/10.1016/j.jmr.2012.12.007DOI Listing
March 2013

Improving the chemical shift dispersion of multidimensional NMR spectra of intrinsically disordered proteins.

J Biomol NMR 2013 Mar 12;55(3):231-7. Epub 2013 Jan 12.

Bruker BioSpin GmbH, 76287 Rheinstetten, Germany.

Intrinsically disordered proteins (IDPs) have recently attracted the attention of the scientific community challenging the well accepted structure-function paradigm. In the characterization of the dynamic features of proteins nuclear magnetic resonance spectroscopy (NMR) is a strategic tool of investigation. However the peculiar properties of IDPs, with the lack of a unique 3D structure and their high flexibility, have a strong impact on NMR observables (low chemical shift dispersion, efficient solvent exchange broadening) and thus on the quality of NMR spectra. Key aspects to be considered in the design of new NMR experiments optimized for the study of IDPs are discussed. A new experiment, based on direct detection of (13)C(α), is proposed.
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http://dx.doi.org/10.1007/s10858-013-9704-3DOI Listing
March 2013

Exclusively heteronuclear (13) C-detected amino-acid-selective NMR experiments for the study of intrinsically disordered proteins (IDPs).

Chembiochem 2012 Nov 11;13(16):2425-32. Epub 2012 Oct 11.

Bruker BioSpin GmbH, 76287 Rheinstetten, Germany.

Carbon-13 direct-detection NMR methods have proved to be very useful for the characterization of intrinsically disordered proteins (IDPs). Here we present a suite of experiments in which amino-acid-selective editing blocks are encoded in CACON- and CANCO-type sequences to give (13) C-detected spectra containing correlations arising from a particular type or group of amino acid(s). These two general types of experiments provide the complementary intra- and inter-residue correlations necessary for sequence-specific assignment of backbone resonance frequencies. We demonstrate the capabilities of these experiments on two IDPs: fully reduced Cox17 and WIP(C) . The proposed approach constitutes an independent strategy to simplify crowded spectra as well as to perform sequence-specific assignment, thereby demonstrating its potential to study IDPs.
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http://dx.doi.org/10.1002/cbic.201200447DOI Listing
November 2012

HMBC-1,1-ADEQUATE via generalized indirect covariance: a high sensitivity alternative to n,1-ADEQUATE.

Magn Reson Chem 2012 Oct 28;50(10):691-5. Epub 2012 Aug 28.

Discovery and Preclinical Sciences-Process & Analytical Chemistry, Merck Research Laboratories, Summit, NJ 07901, USA.

1,1-ADEQUATE and the related long-range 1,n- and n,1-ADEQUATE variants were developed to provide an unequivocal means of establishing (2)J(CH) and the equivalent of (n)J(CH) correlations where n = 3,4. Whereas the 1,1- and 1,n-ADEQUATE experiments have two simultaneous evolution periods that refocus the chemical shift and afford net single quantum evolution for the carbon spins, the n,1-variant has a single evolution period that leaves the carbon spin to be observed at the double quantum frequency. The n,1-ADEQUATE experiment begins with an HMBC-type (n)J(CH) magnetization transfer, which leads to inherently lower sensitivity than the 1,1- and 1,n-ADEQUATE experiments that begin with a (1)J(CH) transfer. These attributes, in tandem, serve to render the n,1-ADEQUATE experiment less generally applicable and more difficult to interpret than the 1,n-ADEQUATE experiment, which can in principle afford the same structural information. Unsymmetrical and generalized indirect covariance processing methods can complement and enhance the structural information encoded in combinations of experiments e.g. HSQC-1,1- or -1,n-ADEQUATE. Another benefit is that covariance processing methods offer the possibility of mathematically combining a higher sensitivity 2D NMR spectrum with for example 1,1- or 1,n-ADEQUATE to improve access to the information content of lower sensitivity congeners. The covariance spectrum also provides a significant enhancement in the F(1) digital resolution. The combination of HMBC and 1,1-ADEQUATE spectra is shown here using strychnine as a model compound to derive structural information inherent to an n,1-ADEQUATE spectrum with higher sensitivity and in a more convenient to interpret single quantum presentation.
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http://dx.doi.org/10.1002/mrc.3863DOI Listing
October 2012

Inversion of 1J(CC) correlations in 1,n-ADEQUATE spectra.

Magn Reson Chem 2012 Aug;50(8):563-8

Merck Research Laboratories, Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structure Elucidation Group, Summit, NJ 07901, USA.

ADEQUATE experiments provide an alternative to the more commonly employed GHMBC experiment for the establishment of long-range heteronuclear connectivities. The 1,1-ADEQUATE experiment allows the unequivocal identification of both protonated and non-protonated carbon resonances adjacent to a protonated carbon. The 1,n-ADEQUATE experiment establishes correlations via an initial (1)J(CH) heteronuclear transfer followed by an (n)J(CC) out-and-back transfer, most typically, via three carbon-carbon bonds. Hence, the 1,n-ADEQUATE experiment allows the equivalent of (4)J(CH) heteronuclear correlations to be probed when they are not observed in a GHMBC spectrum. Aside from the lower sensitivity of the 1,n-ADEQUATE experiment relative to GHMBC experiments, the interpretation of the former is also complicated by the 'leakage' of (1)J(CC) correlations into the spectrum that must be identified. A method for the inversion of (1)J(CC) correlations to facilitate the interpretation of 1,n-ADEQUATE spectra is presented that allows a single experiment to be performed to access (1)J(CC) and (n)J(CC) correlation information.
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http://dx.doi.org/10.1002/mrc.3840DOI Listing
August 2012

Automated protein backbone assignment using the projection-decomposition approach.

J Biomol NMR 2012 Sep 18;54(1):43-51. Epub 2012 Jul 18.

Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, 405 30 Gothenburg, Sweden.

Spectral projection experiments by NMR in conjunction with decomposition analysis have been previously introduced for the backbone assignment of proteins; various pulse sequences as well as the behaviour with low signal-to-noise or chemical shift degeneracy have been illustrated. As a guide for routine applications of this combined tool, we provide here a systematic analysis on different types of proteins using welldefined run-time parameters. As a second result of this study, the backbone assignment module SHABBA was extensively rewritten and improved. Calculations on ubiquitin yielded again fully correct and nearly complete backbone and CHβ assignments. For the 128 residue long azurin, missing assignments mostly affect Hα and Hβ. Among the remaining backbone (plus Cβ) nuclei 97.5 % could be assigned with 1.0 % differences to a reference. Finally, the new SHABBA algorithm was applied to projections recorded for a yeast histone protein domain at room temperature, where the protein is subject to partial unfolding: this leads to unobservable resonances (about a dozen missing signals in a normal 15N-HSQC) and extensive degeneracy among the resonances. From the clearly observable residues, 97.5 % of the backbone and CHβresonances could be assigned, of which only 0.8 % showed differences to published shifts. An additional study on the protein MMP20, which exhibits spectral difficulties to an even larger extent, explores the limitations of the approach.
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http://dx.doi.org/10.1007/s10858-012-9649-yDOI Listing
September 2012

Speeding up sequence specific assignment of IDPs.

J Biomol NMR 2012 Aug 10;53(4):293-301. Epub 2012 Jun 10.

Bruker BioSpin GmbH, Silberstreifen, 76287 Rheinstetten, Germany.

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct (13)C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.
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http://dx.doi.org/10.1007/s10858-012-9639-0DOI Listing
August 2012

Structural characterisation of a histone domain by projection-decomposition.

J Magn Reson 2012 Apr 23;217:48-52. Epub 2012 Feb 23.

Biophysics Group, Department of Chemistry, University of Gothenburg, Box 462, 405 30 Gothenburg, Sweden.

We demonstrate that two projection experiments, a (15)N-HSQC-NOESY-(15)N-HSQC and a (13)C-HSQC-NOESY-(15)N-HSQC, recorded for a histone domain from yeast, contain enough information to support a structural characterisation of the protein. At the temperature used, 298 K, the histone domain exhibits a very high extent of chemical shift degeneracy that is uncharacteristic for a fully folded domain. Nonetheless, a structured core of 67 residues, which is formed by three α-helices and a two-stranded β-sheet is defined by this NOESY data; this core structure was shown earlier to be present at lower temperature. The above two experiments, which together required 18 h of instrument time, are part of a set of five projection experiments acquired during 2.5 days with the goal of complete characterisation of proteins, including full resonance assignment and structure.
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http://dx.doi.org/10.1016/j.jmr.2012.02.006DOI Listing
April 2012

Speeding up the measurement of one-bond scalar (1J) and residual dipolar couplings (1D) by using non-uniform sampling (NUS).

J Magn Reson 2012 Mar 1;216:134-43. Epub 2012 Feb 1.

Clemens Schöpf Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Petersenstr. 22, 64287 Darmstadt, Germany.

The accurate and precise measurement of one-bond scalar and residual dipolar coupling (RDC) constants is of prime importance to be able to use RDCs for structure determination. If coupling constants are to be extracted from the indirect dimension of HSQC spectra a significant saving of measurement time can be achieved by non-uniform sampling (NUS). Coupling constants can either be obtained with the same precision as in traditionally acquired spectra in a fraction of the measurement time or the precision can be significantly improved if the same amount of measurement time as for traditionally acquired spectra is invested. The application of NUS for the measurement of (1)J (scalar coupling constants) and (1)T (total couplings constants) from different kinds of ω(1)-coupled spectra (including also J-scaled ones) is examined in detail and the possible gains in time or resolution are discussed. When using the newly proposed compressed sensing (CS) algorithm for processing, the quality of the spectra is comparable to the traditionally sampled ones.
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http://dx.doi.org/10.1016/j.jmr.2012.01.008DOI Listing
March 2012

New strategies for designing robust universal rotation pulses: application to broadband refocusing at low power.

J Magn Reson 2012 Mar 26;216:78-87. Epub 2012 Jan 26.

Physics Department, Wright State University, Dayton, OH 45435, USA.

Optimizing pulse performance often requires a compromise between maximizing signal amplitude and minimizing spectral phase errors. We consider methods for the de novo design of universal rotation pulses, applied specifically but not limited to refocusing pulses. Broadband inversion pulses that rotate all magnetization components 180° about a given fixed axis are necessary for refocusing and mixing in high-resolution NMR spectroscopy. The relative merits of various methodologies for generating pulses suitable for broadband refocusing are considered. The de novo design of 180° universal rotation pulses (180(UR)(°)) using optimal control can provide improved performance compared to schemes which construct refocusing pulses as composites of existing pulses. The advantages of broadband universal rotation by optimized pulses (BURBOP) are most evident for pulse design that includes tolerance to RF inhomogeneity or miscalibration. Nearly ideal refocusing is possible over a resonance offset range of ± 170% relative to the nominal pulse B(1) field, concurrent with tolerance to B(1) inhomogeneity/miscalibration of ± 33%. We present new modifications of the optimal control algorithm that incorporate symmetry principles (S-BURBOP) and relax conservative limits on peak RF pulse amplitude for short time periods that pose no threat to the probe. We apply them to generate a set of low-power 180(BURBOP)(°) pulses suitable for widespread use in (13)C spectroscopy on the majority of available probes. A quantitative measure for the reduced spectral phase error provided by these symmetry principles is also derived. For pulses designed according to this symmetry, refocusing phase errors are virtually eliminated upon application of EXORCYCLE or an equivalent G-180(S-BURBOP)(°)-G gradient sandwich, independent of resonance offset and RF inhomogeneity. The magnitude of the refocused component is not significantly compromised in achieving such ideal phase performance.
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http://dx.doi.org/10.1016/j.jmr.2012.01.005DOI Listing
March 2012

13C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides.

J Biomol NMR 2010 Aug 11;47(4):259-69. Epub 2010 Jun 11.

Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University Frankfurt, Frankfurt/M, Germany.

We present here a set of (13)C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose (13)C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4' nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1',H1' ribose signals. The experiments were applied to two RNA hairpin structures. The current set of (13)C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, (13)C-direct detected NMR methods constitute useful complements to the conventional (1)H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.
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http://dx.doi.org/10.1007/s10858-010-9429-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2900595PMC
August 2010

A non-uniformly sampled 4D HCC(CO)NH-TOCSY experiment processed using maximum entropy for rapid protein sidechain assignment.

J Magn Reson 2010 May 1;204(1):160-4. Epub 2010 Mar 1.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, 4072 QLD, Australia.

One of the stiffest challenges in structural studies of proteins using NMR is the assignment of sidechain resonances. Typically, a panel of lengthy 3D experiments are acquired in order to establish connectivities and resolve ambiguities due to overlap. We demonstrate that these experiments can be replaced by a single 4D experiment that is time-efficient, yields excellent resolution, and captures unique carbon-proton connectivity information. The approach is made practical by the use of non-uniform sampling in the three indirect time dimensions and maximum entropy reconstruction of the corresponding 3D frequency spectrum. This 4D method will facilitate automated resonance assignment procedures and it should be particularly beneficial for increasing throughput in NMR-based structural genomics initiatives.
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http://dx.doi.org/10.1016/j.jmr.2010.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859963PMC
May 2010

Exclusively heteronuclear NMR experiments to obtain structural and dynamic information on proteins.

Chemphyschem 2010 Feb;11(3):689-95

Bruker BioSpin GmbH, Silberstreifen, 76287 Rheinstetten, Germany.

Provided that (13)C-detected NMR experiments are either preferable or complementary to (1)H detection, we report here tools to determine C(alpha)-C', C'-N, and C(alpha)-H(alpha) residual dipolar couplings on the basis of the CON experiment. The coupling constants determined on ubiquitin are consistent with the subset measured with the (1)H-detected HNCO sequences. Since the utilization of residual dipolar couplings may depend on the mobility of the involved nuclei, we also provide tools to measure longitudinal and transverse relaxation rates of N and C'. This new set of experiments is a further development of a whole strategy based on (13)C direct-detection NMR spectroscopy for the study of biological macromolecules.
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http://dx.doi.org/10.1002/cphc.200900772DOI Listing
February 2010

Speeding up (13)C direct detection biomolecular NMR spectroscopy.

J Am Chem Soc 2009 Oct;131(42):15339-45

Bruker BioSpin GmbH, Silberstreifen, 76287 Rheinstetten, Germany.

After the exploitation of (1)H polarization as a starting source for (13)C direct detection experiments, pulse sequences are designed which exploit the accelerated (1)H longitudinal relaxation to expedite (13)C direct detection experiments. We show here that 2D experiments based on (13)C direct detection on a 0.5 mM water sample of ubiquitin can be recorded in a few minutes and 3D experiments in a few hours. We also show that fast methods like nonuniform sampling can be easily implemented. As overall experimental time has always been a counter indication for the use of (13)C direct detection experiments, this research opens new avenues for the application of (13)C NMR to biological molecules.
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http://dx.doi.org/10.1021/ja9058525DOI Listing
October 2009