Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients

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Introduction

During the last decade a variety of triple resonance pulse sequences that are used for the structure determination of proteins by heteronuclear multidimensional NMR spectroscopy have been introduced and optimized. Here, we would like to give a critical review of experiments that have been proposed for chemical shift assignment and for the extraction of distance restraints based on NOE experiments. Detailed descriptions of useful implementations of pulse sequences will be given with respect to improvements in signal-to-noise ratio and artifact reduction, especially by the use of pulsed field gradients in combination with sensitivity enhancement and water-flip-back schemes.

The introduction of three- and four-dimensional NMR experiments [1], [2], [3], [4], [5] and the availability of 13C-,15N-labelled proteins [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16] allow one to assign the proton, nitrogen and carbon chemical shifts of proteins and protein complexes with molecular weights well above 25 kD [17], [18] and to determine their structures in solution [19], [20], [21], [22]. The resonance assignment of singly (15N or 13C) labelled proteins using 3D experiments [23] is basically an extension of Wüthrich’s strategy which exclusively relies on homonuclear 1H NMR experiments [24]. The conventional assignment strategies for unlabelled or singly labelled molecules make combined use of experiments with coherent (COSY, TOCSY) and incoherent (NOESY, ROESY) magnetization transfer. However, incoherent magnetization transfer using the dipolar interaction is dependent on the secondary structure, which can cause misassignments, especially in crowded spectra. The introduction of heteronuclear triple resonance experiments relying exclusively on 1J/2J couplings, which are to first order independent of conformation [9], [25] triggered a development that to date has yielded a large number of experiments for 13C-, 15N-labelled proteins and new assignment strategies. As 1J/2J couplings are relatively large, they yield fast coherence transfers that can compete with the loss of magnetization as a result of relaxation during the pulse sequence. This becomes more and more important with increasing molecular weight. Recently, the use of deuterium labellling has been shown to increase the relaxation times dramatically, thereby allowing the study of proteins with molecular weights well above 30 kD [26], [27].

The intention of this review is to summarize triple resonance pulse sequences that have been introduced and to evaluate their performance and applicability with respect to the molecular weight of the protein to be studied. Useful implementations of these experiments based on the practical experience of the authors are presented in greater detail. Further, experimental aspects and optimizations of the pulse sequences are discussed. We will especially focus on the use of pulsed field gradients in combination with sensitivity-enhanced magnetization transfers and water-flip-back schemes to improve the quality of the NMR spectra.

In Section 2, the various assignment strategies that depend on the respective isotope labellling scheme are introduced. In Section 3, basic building blocks and experimental aspects of the implementation of multidimensional triple resonance experiments are discussed. Pulse sequences for the assignment of backbone (Section 4) and side chain resonances (Section 5) in 13C-,15N-labelled proteins are described, thereafter including modified pulse sequences for deuterated samples. 3D and 4D NOESY experiments used to derive internuclear distance restraints for structure calculations are discussed in Section 6. For applications on complexes (e.g. between an unlabelled peptide and a 13C-, 15N-labelled protein) these experiments can be modified by the use of spectral editing and filter elements in order to separate inter- and intramolecular NOEs (Section 6.2).

We would also like to direct the reader’s attention to some recent developments which are not discussed in this review but are expected to have a great impact in the field of biomolecular NMR spectroscopy in the near future. In the context of studying protein/ligand interactions we would like to refer to a very elegant and efficient technique that is used for identifying and designing high-affinity binding ligands for proteins in pharmaceutical research. The method called “SAR by NMR” (Structure Activity Relationship by NMR) is based on monitoring chemical shift changes of amide protons and nitrogens which accompany the titration of potential ligands to a solution of 15N-labelled protein [28], [29].

More recently, some promising new methodological developments have been introduced. These refer to novel types of structural restraints that have been investigated for use in high-resolution NMR studies of biomolecules. One type of restraints is based on residual dipolar couplings that are observed if a small alignment of the biomolecule can be achieved in solution [30], [31], [32]. The angle- and distance dependence of these residual dipolar couplings provide true long range structural restraints. Thus, if used in combination with NOE-based distance restraints these should significantly increase the precision and accuracy of NMR-derived structures. Another type of experiment exploits the angular dependence of cross-correlated relaxation, i.e. dipole/dipole or dipole/CSA [33]. In these experiments direct projections of bond vectors onto each other are obtained without the need for calibration, as is required for angular restraints derived from J-couplings. These projection restraints should therefore improve structure determination in a similar way as the orientational restraints derived from residual dipolar couplings. Moreover, since the novel long range restraints not only supplement but may also partially replace NOE based distance restraints, it is conceivable that – in combination with 2H-labelling schemes – this will allow the determination of three-dimensional structures for proteins with molecular weights well above 50 kD.

In addition, NMR techniques, called Transverse Relaxation-Optimized Spectroscopy (TROSY) [34], have recently been introduced that largely attenuate T2-relaxation at high magnetic fields by mutual cancellation of dipole/dipole and CSA relaxation mechanisms. Similar effects have been observed by removing the heteronuclear dipolar relaxation through multiple quantum line narrowing (see Section 3.6). Combination of these principles with other strategies, i.e. deuteration and the aforementioned novel structural restraints, should provide an avenue to successful NMR structure determination for very large biological macromolecules with molecular weights well above 50 kD.

Section snippets

Strategies and experiments for the resonance assignment of uniformly 13C-, 15N-labelled proteins

The assignment strategy for proteins that are not isotopically enriched [24] makes use of a combination of COSY/TOCSY and NOESY or ROESY spectra. The spin systems attached to each HN are identified in the COSY and TOCSY spectra. NOESY or ROESY are used for the sequential assignment of the individual spin systems. The establishing of the sequential connectivity relies on the occurrence of the resonance frequency of the Hα(i) proton of amino acid (i), which is observed in the HN(i), Hα(i) cross

Basic tools and experimental aspects of triple resonance pulse sequences

The NMR pulse sequences for the assignment of uniformly 13C-, 15N-labelled proteins are composed of a large number of pulses that have to be applied at three or four different frequencies in order to excite 1H, 15N, aliphatic 13C and 13C′ resonances. As signal losses as a result of miscalibration or pulse imperfections accumulate, accurate calibration of pulses and good RF homogeneity of the probe on all frequency channels is essential. The aliphatic and carbonyl pulses can either be generated

Experiments for the assignment of backbone resonances

In the following two sections, recommended pulse sequences for the assignment of backbone and side chain resonances using uniformly 13C,15N-labelled proteins will be discussed. Where appropriate, sensitivity-enhanced back transfer is combined with a heteronuclear gradient echo and water-flip-back. The use of these building blocks yields excellent solvent suppression without saturation of exchangeable proton signals. This is of prime importance since most of the experiments described excite

Combined backbone and side chain assignment

In principle, the experiments described in the previous section can yield complete backbone assignments and establish the sequential connectivities. However, overlap even in 3D spectra results in ambiguities. Therefore, an additional assignment strategy has been developed based on the use of the chemical shifts of the Cα,β and Hα,β resonances for the sequential assignment. As the chemical shifts of the side chain carbons are characteristic for the amino acid type, this information can also be

3D and 4D NOESY/ROESY experiments

After the assignment of all or nearly all resonances of a protein, experiments for the extraction of structural parameters are analyzed. The most important parameter for NMR-based structure determination are 1H,1H distances which are derived from NOE intensities, and dihedral angles which are obtained from 3J coupling constants. In this section, we will describe 13C- and 15N-edited NOESY experiments that are used for the measurement of NOE intensities, and experiments for the extraction of

Conclusions

The intention of this review was to summarize and evaluate the numerous triple resonance pulse sequences that have been introduced since 1989 for the assignment of doubly (13C, 15N) or triply (13C, 15N, 2H)-labelled proteins and the extraction of intra- and intermolecular distance restraints from NOESY experiments. Special attention has been given to the detailed discussion of optimized pulse sequences rather than to list all possible implementations without comment. A typical set of NMR

Acknowledgements

This work was supported by the Fonds der Chemischen Industrie and the DFG and is mainly based on the results of two Ph.D. theses (M.S. and J.S.). We would like to thank numerous people for valuable discussions and contributions to this article: Dr. H. Schwalbe, M. Maurer, PD Dr. S. Glaser, Dr. T. Carlomagno, Dr. H. Försterling (University Frankfurt, Germany); Dr. E.T. Olejniczak, Dr. L. Yu, Dr. S.W. Fesik (Abbott Labs, USA), Dr. H. Kovacs (EMBL Heidelberg, Germany). We would also like to thank

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References (246)

  • C. Griesinger et al.

    J. Magn. Reson.

    (1987)
  • G.W. Vuister et al.

    J. Magn. Reson.

    (1988)
  • C. Griesinger et al.

    J. Magn. Reson.

    (1989)
  • D.C. Muchmore et al.

    Methods Enzymol.

    (1989)
  • S.W. Fesik et al.

    J. Magn Reson.

    (1988)
  • S.S. Wijmenga et al.

    J. Magn. Reson.

    (1989)
  • K. Shon et al.

    J. Magn. Reson.

    (1989)
  • M. Ikura et al.

    J. Magn. Reson.

    (1990)
  • E.R.P. Zuiderweg et al.

    J. Magn. Reson.

    (1990)
  • S.J. Archer et al.

    J. Magn. Reson.

    (1991)
  • L.E. Kay et al.

    J. Magn. Reson.

    (1990)
  • L.E. Kay et al.

    J. Magn. Reson.

    (1991)
  • R. Powers et al.

    J. Magn. Reson.

    (1991)
  • S. Grzesiek et al.

    J. Magn. Reson.

    (1992)
  • L.E. Kay et al.

    J. Magn. Reson.

    (1992)
  • L.E. Kay et al.

    J. Magn. Reson.

    (1991)
  • R.T. Clubb et al.

    J. Magn. Reson.

    (1992)
  • J. Engelke et al.

    .J. Magn. Reson.

    (1995)
  • S. Grzesiek et al.

    J. Magn. Reson.

    (1992)
  • M. Wittekind et al.

    J. Magn. Reson.

    (1993)
  • S. Grzesiek et al.

    J. Magn. Reson.

    (1993)
  • B.A. Lyons et al.

    J. Magn. Reson.

    (1993)
  • J.M. Richardson et al.

    J. Magn. Reson.

    (1993)
  • A. Bax et al.

    J. Magn. Reson.

    (1990)
  • A. Bax et al.

    J. Magn. Reson.

    (1990)
  • T. Szyperski et al.

    J. Magn. Reson

    (1994)
  • B. Brutscher et al.

    J. Magn. Reson.

    (1994)
  • M. Wittekind et al.

    J. Magn. Reson.

    (1993)
  • K. Gehring et al.

    J. Magn. Reson.

    (1995)
  • J.L. Sudmeier et al.

    J. Magn. Reson.

    (1996)
  • M. Pellecchia et al.

    J. Magn. Reson.

    (1997)
  • V. Dötsch et al.

    J. Magn. Reson.

    (1996)
  • N.S. Rao et al.

    J. Magn. Reson.

    (1996)
  • L. Szilagyi et al.

    J. Magn. Reson.

    (1989)
  • A. Pastore et al.

    J. Magn. Reson.

    (1990)
  • D.S. Wishart et al.

    J. Mol. Biol.

    (1991)
  • L.E. Kay et al.

    Science

    (1990)
  • R.R. Ernst

    Angew. Chem.

    (1992)
  • L.P. McIntosh et al.

    Proc. Natl. Acad. Sci.

    (1984)
  • R.H. Griffey et al.

    J. Am. Chem. Soc.

    (1986)
  • L.P. McIntosh et al.

    J. Biomol. Struct. Dyn.

    (1987)
  • B.H. Oh et al.

    Science

    (1988)
  • W.M. Westler et al.

    J. Am. Chem. Soc.

    (1988)
  • B.J. Stockman et al.

    Biochemistry

    (1989)
  • R.A. Venters et al.

    Biochemistry

    (1991)
  • A.P. Hansen et al.

    Biochemistry

    (1992)
  • T. Kigawa et al.

    J. Biomol. NMR

    (1995)
  • J.W. Lustbader et al.

    J. Biomol. NMR

    (1996)
  • R.H. Fogh et al.

    J. Biomol. NMR

    (1994)
  • M.L. Remerowski et al.

    J. Biomol. NMR

    (1994)
  • Cited by (0)

    View full text