Four-dimensional NMR

For 20 years center stage has been occupied by two-dimensional (and now three-and four-dimensional) NMR techniques. 2D NMR and its offshoots offer two distinct advantages (1) relief from overcrowding of resonance lines, as the spectral information is spread out in a plane or a cube rather than along a single frequency dimension, and (2) opportunity to correlate pairs of resonances. In the latter respect 2D NMR has features in common with various double resonance methods, but as we shall see, 2D NMR is far more efficient and versatile. Hundreds of different 2D NMR techniques have been proposed in the literature, but most of these experiments can be considered as variations on a rather small number of basic approaches. Once we develop familiarity with the basic principles, it will be relatively easy to understand most variations of the standard 2D experiments. 

The ability to manipulate spins in two-dimensional experiments and to transfer magnetization between spins has made it possible to use a sensitive nucleus (primarily H) to measure the spectral features of less sensitive nuclei, such as 13C and 15N. Several methods are commonly used, but each begins with a H pulse sequence, often resembling the one in INEPT (Section 9.7). As in INEPT, a combination of H and X pulses transfers polarization to the X spin system. In some instances further transfers are made to another spin system (Y), then back through X to H, where the signal is detected. Thus, the large polarization of the proton is used as the basis for the experiment, and the high sensitivity of H NMR is exploited for detection. Such indirect detection methods use two-, three-, and sometimes four-dimensional NMR. 

Kupce E, Freeman R (2004) Fast reconstruction of four-dimensional NMR spectra from plane projections. J Biomol NMR 28 391-395... 

Tugarinov V, Muhandiram R, Ayed A et al (2002) Four-dimensional NMR spectroscopy of a 723-residue protein chemical shift assignments and secondary structure of malate synthase G. J Am Chem Soc 124 10025-10035... 

Yang D, Kay LE (1999) TROSY triple-resonance four-dimensional NMR spectroscopy of a 46 ns tumbling protein. J Am Chem Soc 121 2571-2575... 

Three and four-dimensional NMRs are also available. They are more complicated than one- and two-dimensional NMR, but the underlying general principles are about the same. The three- and four-dimensional NMRs are useful for the determination of stmctmes of larger proteins and protein complexes. 

In the following, we will discuss heteronuclear polarization-transfer techniques in four different contexts. They can be used as a polarization-transfer method to increase the sensitivity of a nucleus and to shorten the recycle delay of an experiment as it is widely used in 1H-13C or 1H-15N cross polarization. Heteronuclear polarization-transfer methods can also be used as the correlation mechanism in a multi-dimensional NMR experiment where, for example, the chemical shifts of two different spins are correlated. The third application is in measuring dipolar coupling constants in order to obtain distance information between selected nuclei as is often done in the REDOR experiment. Finally, heteronuclear polarization transfer also plays a role in measuring dihedral angles by generating heteronuclear double-quantum coherences. 

Fig. 7. One-dimensional NMR spectra of the designed four-helix bundles SA-42 (lower trace) and GTD-43 (top two traces). The chemical shift dispersion of SA-42 in 90% H2O and 10% D2O at 323 K and pH 4.5 is poor and the resonances are severely broadened due to conformational exchange. The chemical shift dispersion of GTD-43 in the same solvent at 288 K and pH 3.0 is comparable to that of the naturally occurring four-helix bundle IL-4 and the resonances are not significantly affected by conformational exchange. Upon raising the temperature to 298 K line broadening is observed (top trace) which shows that GTD-43 is in slow exchange on the NMR time scale, unlike SA-42 where an increased temperature reduces the line width. These spectra are therefore diagnostic of structures with disordered (SA-42) and ordered (GTD-43) hydrophobic cores...

Figure 9.1 represents a one-dimensional NMR spectrum (the intensities of the peaks are not considered to be a second dimension). More sophisticated NMR studies, in two, three or four dimensions can be used to determine the position of ail the atoms present in a molecule. This chapter only deals with one-dimensional (1 -D) NMR. 

Builder, S.E. and W.S. Hancock Analytical and Process Chromatography in Pharmaceutical Protein Production, Chem. Eng. Progress, 42 (August 1988). Clore, G.M, and A M. Gronenbom Structures of Larger Proteins in Solution Three- and Four-Dimensional Heteronuclear NMR Spectroscopy." Science, 1390 (June 7. 1991). 

Clore, G.M. Gronenbom, A.M. (1991). Structures of larger proteins in solution Three- and four-dimensional heteronuclear NMR spectroscopy. Science 252, 1390-1399. 

In this chapter, using these four compounds as examples, we turn our attention to correlation NMR spectrometry most (but not all) of the useful experiments fall into the category of two-dimensional NMR. Our approach in this chapter is to present the spectra for each compound independently as a logical set. Most of the general aspects of each experiment are given in the discussion of ipsenol others are only introduced with the more complicated compounds. The material for ipsenol should be thoroughly covered first. The other compounds can be covered independently or not at all. 

Four-dimensional exchange NMR was introduced some time ago for examining the correlations between different molecular motions.53 The... 

In 1971, the idea of 2D NMR spectroscopy was proposed by Jeener and later implemented by Aue, Bartholdi and Ernst, who published their work in 1976.47 The first experiments, carried out mostly in the liquid phase, have unambiguously proved that 2D NMR spectra provide more information about a molecule than ID NMR spectroscopy and are especially useful in determining the structure of molecules that are too complicated to work with using ID NMR. With the progress in the methodology and software improvement, three-dimensional (3D) and four-dimensional (4D) NMR experiments were gradually introduced into the laboratory practice. Such strategy, the so-called multi-dimensional (or ND) NMR spectroscopy, has found a number of spectacular applications in the structure analysis of natural products. 

G. M. Clore and A. M. Gronenborn, Prog. NMR Spectrosc., 23, 43 (1991). Applications of Three- and Four-Dimensional Heteronudear NMR Spectroscopy to Protein Structure Determination. 

FIGURE 10.1 Schematic representation of a basic two-dimensional NMR experiment in terms of four periods preparation, rP evolution, lt mixing, rM detection, t2. For a given experiment, rP and rM are usually fixed periods, while I, and t2 are variable time periods, as described in the text. 

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