Three-dimensional NMR Spectra Research Articles

Quantitation of Resonances in Biological 31P NMR Spectra via Principal Component Analysis: Potential and Limitations

This paper examines the potential and limitations of peak area quantitation of biological NMR spectra using principal component analysis (PCA), including its requirement for prior knowledge. The principles of the method are presented without in-depth mathematical treatment. PCA is illustrated for simulated data, 31P NMR spectra obtained consecutively over 1–2.5 days from perfused Rat-2 cells metabolizing the choline analogue phosphoniumcholine (Chop) and in vivo proton-decoupled, NOE-enhanced, three-dimensional CSI localized 31P NMR spectra of the liver of healthy volunteers. The results show that PCA can be used to quantitate strongly overlapping peaks without prior knowledge of the peak shapes or positions and to reconstruct spectra with significantly reduced noise variance. Two major limitations of PCA are presented: (1) PCA cannot separate peaks whose intensities are well correlated; (2) PCA is sensitive to differences in chemical shift and line-width of peaks between spectra. The discussion focusses on what knowledge of the biological and spectroscopic features of the samples and the principles of PCA is necessary for peak area quantitation via PCA.

Experimental Aspects of Multidimensional Exchange Solid-State NMR

A five-pulse sequence for two-dimensional exchange 2H NMR that results in improved processability of the obtained spectra is presented, together with the phase cycle necessary to eliminate unwanted signal contributions. The scheme of the three-dimensional Fourier transformation to yield pure-absorption-mode three-dimensional 2H NMR spectra is presented, and the off-resonance measurement of multidimensional 2H NMR experiments is discussed. Furthermore, the phase cycles of six- and eight-pulse sequences for the acquisition of undistorted three-dimensional 2H NMR spectra are derived from the phase cycles of the 2D pulse sequences. Differences in the phase cycles for spin-12 nuclei, such as 13C, and spin-1 nuclei, such as 2H, are explicitly addressed.

1H, 13C, and 15N assignments and secondary structure of the FK506 binding protein when bound to ascomycin.

The 1H, 13C, and 15N resonances of FKBP when bound to the immunosuppressant, ascomycin, were assigned using a computer-aided analysis of heteronuclear double and triple resonance three-dimensional nmr spectra of [U-15N]FKBP/ascomycin and [U-15N,13C]FKBP/ascomycin. In addition, from a preliminary analysis of two heteronuclear four-dimensional data sets, 3JHN,H alpha coupling constants, amide exchange data, and the differences between the C alpha and C beta chemical shifts of FKBP to random coil values, the secondary structure of FKBP when bound to ascomycin was determined. The secondary structure of FKBP when bound to ascomycin in solution closely resembled the x-ray structure of the FKBP/FK506 complex but differed in some aspects from the structure of uncomplexed FKBP in solution.

Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.

Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy.

We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on 15N-enriched and 13C/15N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem, 203, 483-491] to the side-chain 1H,15N and 13C resonances. From both 3D heteronuclear 1H-NOE 1H-13C and 1H-NOE 1H-15N multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the C alpha atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl, Acad. Sci. USA 89, 2499-2503).

Unraveling overlapping multiplets in two-dimensional NMR spectra by selective injection of coherence

In order to separate overlapping diagonal multiplets in two- or three-dimensional NMR spectra, new sequences using selective pulses are described which allow one to “inject” magnetization at the beginning of the evolution period by transfer from a selected coupling partner. The techniques (dubbed MUSIC, for multiplet unraveling by selective injection of coherence) can be adapted for the separation of overlapping cross peaks.

Phasing two- and three-dimensional NMR spectra by use of the Hilbert transform can save computer time and space

Use of the Hilbert transformation of phasing reduces disk space requirements and leads to considerably faster processing rates for two-dimensional and particularly three-dimensional NMR spectra. The reduction in storage space is a consequence of the need to store only the real part of the spectrum. The decreased processing time results primarily from the reduction in the number of disk input/output steps. Striking time savings have been documented with software operating on several different computers.

Heteronuclear 3D NMR and isotopic labeling of calmodulin: Towards the complete assignment of the 1H NMR spectrum

New methods are described that permit detailed analysis of the NMR spectra of calmodulin, an α-helical protein with a molecular weight of 16.7 kD. Two complementary approaches have been used: uniform labeling with 15N and labeling of specific amino acids with either 15N or 13C. It is demonstrated that uniform 15N labeling permits the recording of sensitive three-dimensional (3D) NMR spectra that show far better resolution than their conventional two-dimensional analogs. Selective 15N labeling of amino acids can be used for identifying the type of amino acid, providing information that is essential for the analysis of the 3D spectra. Simultaneous selective labeling with both 15N and 13C can provide a number of unique backbone assignments from which sequential assignment can be continued.

Assignment strategies in homonuclear three-dimensional proton NMR spectra of proteins

The increase in dimensionality of three-dimensional (3D) NMR greatly enhances the spectral resolution in comparison to 2D NMR. It alleviates the problem of resonance overlap and may extend the range of molecules amenable to structure determination by high-resolution NRM spectroscopy. Here, we present strategies for the assignment of protein resonances from homonuclear nonselective 3D NOE-HOHAHA spectra. A notation for connectivities between protons, corresponding to cross peaks in 3D spectra, is introduced. We show how spin systems can be identified by tracing cross-peak patterns in cross sections perpendicular to the three frequency axes. The observable 3D sequential connectivities in proteins are tabulated, and estimates for the relative intensities of the corresponding cross peaks are given for alpha-helical and beta-sheet conformations. Intensities of the cross peaks in the 3D spectrum of pike III parvalbumin follow the predictions. The sequential-assignment procedure is illustrated for loop regions, extended and alpha-helical conformations for the residues Ala 54-Leu 63 of parvalbumin. NOEs that were not previously identified in 2D spectra of parvalbumin due to overlap are found.

Local symmetry in 2D and 3D NMR spectra

Symmetry properties of peaks in two- and three-dimensional NMR spectra are described by irreducible representations of point symmetry groups. The symmetry of peaks is derived from factorizations in terms of basic patterns and lower dimensional spectra. Procedures are indicated for the symmetry decomposition of peak multiplets.