Multidimensional heteronuclear NMR experiment

Multidimensional heteronuclear NMR experiments for determining the structure of isotopically labelled RNA were discussed in detail by Pardi. ... 

Sattler M, Schleucher J, Griesinger C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Progress Nuclear Magnet. Reson. Spectros. 1999 34 93-158. 

In order to pursue heteronuclear multidimensional NMR experiments, a bacterial system for expression of apoLp-III has been developed which allows facile production of 150 - 200 mg/L l N-iabeled apoLp-III or 100 - 125 mg/L 15N/l3c-double labeled apoLp-III. Figure 2, panel A shows the IH- n HSQC spectrum of a 1.0 mM solution of lipid-free, uniformly N-labeled apoLp-III. Panel A also indicates that, although the chemical shift dispersion in the H-dimension is rather small (6.5 ppm to 9.5 ppm), it is generally upfield shifted, consistent with the fact that the protein secondary structure is predominantly a-helix (13). The chemical shifts in the N-dimension are well-dispersed which results in good separation of the overall crosspeaks. However, certain regions in the spectrum are still crowded as shown in Figure 2. 

NMR has been a powerful technique for structural analyses of macromolecules. However, ID NMR spectra of PDMS are usually complicated due to signal overly. Their complete characterization often requires combinations of several techniques. Multidimensional NMR techniques, especially inversely detected 3D heteronuclear shift correlation experiments, offer the opportunity to obtain the complete structural characterization by using NMR experiments alone. Biological 3D-NMR experiments are usually performed in conjunction with uniform and isotopic labeling. In polymer chemistry, when isotopic labeling is possible, it is often very difficult and expensive. By modifying the 3D-pulse sequence used for biopolymers, triple resonance 3D-NMR techniques have been adapted for sbufying the structures of polymers, which involve H- C- P, H- C- Si spin... 

There have been many multidimensional NMR experiments published for homonuclear H- H studies and for H-heteronuclear studies as well as others involving heteronuclei only. Many are variations of general schemes for specific applications and only a few 2D NMR experiments have become widely used. The following sections discuss these principal 2D NMR experiments and the reader is referred to the Further Reading section for specific details of the theory and practical aspects of multidimensional NMR. 

During the multidimensional NMR experiments, effective coherence transfer is important, which can be accomplished via accurate J-modulation (e.g.. Insensitive Nuclei Enhanced by Polarization Transfer, INEPT) or dipole-dipole (Nuclear Over-hauser Effect, NOE) interaction. As seen in the table, the couplings with F are quite different from those with H and The large and varied F couplings create opportunities and permit a larger variety of experiments, while at the same time, it also create complicated spectra with complex cross-peak patterns. The Vcf and Jcp in Table 24.2 is sufficient for the heteronuclear 2D-NMR experiments... 

Multidimensional solid-state NMR experiments have been shown to yield completely resolved spectra of uniformly labelled proteins in oriented lipid bilayers. In three-dimensional spectra, each amide resonance is characterized by three frequencies ( H chemical shift, chemical shift and H-i N heteronuclear dipolar coupling), which provide the source of resolution among the various sites as well as the basic input for structure determination based on orientational constraints. The data shown in Figure 5 are from a 50-residue protein in oriented lipid bilayers. More importantly, since the polypeptides are immobilized by the lipids on the relevant NMR time-scales, there can be no further degradation of line widths or other spectroscopic properties as the size of the polypeptide increases. Although larger proteins will have more complex spectra resulting from the increased number of resonances, there is no fundamental size limitation to solid-state NMR studies of membrane proteins. 

Cross polarization can also be used for magnetization transfer between low-gamma nuclei like and as a mixing sequence in a multidimensional NMR experiment. Due to the substantial shift separation of about 120 ppm between carbonyl and Ca carbon atoms in proteins, the CP transfer can be tailored specifically to NCa- or NCO-transfer (SPECIFIC-CP) [81]. In protein NMR spectroscopy such a heteronuclear correlation experiment may be incorporated into multidimensional experiments and thus be used to residue-specific resonance assignment in NMR spectroscopy of immobilized peptides or proteins [82]. 

A simple way of illustrating multidimensional NMR is through reference to hetero-nuclear correlation spectroscopy, in which two or more separate frequency dimensions are correlated with one another. For example, a particularly valuable 2D experiment is heteronuclear single quantum correlation (HSQC) spectroscopy, in which the resultant spectrum has two frequency axes, corresponding to and frequency dimensions, and one intensity axis. Analogous HSQC... 

The introduction and implementation of heteronuclear-based multidimensional techniques have revolutionized the protein NMR field. Large proteins (> 100 residues) are now amenable to detailed NMR studies and structure determination. These techniques, however, necessarily require a scheme by which and isotopes can be incorporated into the protein to yield a uniformly labeled sample. Additional complications, such as extensive covalent post-translational modifications, can seriously limit the ability to efficiently and cost effectively express a protein in isotope enriched media - the c-type cytochromes are an example of such a limitation. In the absence of an effective labeling protocol, one must therefore rely on more traditional proton homonuclear NMR methods. These include two-dimensional (1) and, more recently, three-dimensional H experiments (2,3). Cytochrome c has become a paradigm for protein folding and electron transfer studies because of its stability, solubility and ease of preparation. As a result, several high-resolution X-ray crystal structure models for c-type cytochromes, in both redox states, have emerged. Although only subtle structural differences between redox states have been observed in these... 

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