Triple-resonance NMR

Considering that the literature on the development of experimental methods and important fields of application of X/Y correlations in inorganic, organoelement and organometallic chemistry up to 1997 has been covered in earlier reviews,11 we will focus here on recent improvements of experimental techniques and novel applications for compound characterisation. Despite the recently increasing interest in the application of X/Y correlation spectroscopy in solids,12,13 this review will cover only solution NMR techniques. Likewise, a survey of specialised triple-resonance NMR experiments devoted to the characterisation of bio-molecules, and their application, is considered beyond the scope of this article. 

As would be expected, several additional applications of cryoprobe technology in the area of biomacromolecular NMR were also published. Goger et al,253 described the reduction in time required for the acquisition of triple resonance NMR experiments performed on proteins. Later in the year, Craik et al.254 reported a study of the 21 amino acid bacterial peptide microcin J25 that relied on cryoprobe technology. In late 2003, Bertini and co-workers255 reported the application of cryoprobe-based 13C direct-detection NMR experiments on a paramagnetic oxidized superoxide dismutase. 

An interesting example is calmodulin, which consists of two domains. The two domains are connected by a long a-helical linker. When the structure was first determined by X-ray crystallography in 1984, it showed an intriguing dumbbell shape. It was a controversial result because many questioned whether the linker helix really existed in solution too or was only an artifact of crystallization. We used calmodulin to develop many of what is known today as triple-resonance NMR techniques [Ikura, M. Kay, L. E. Bax, A. Biochemistry 1990, 29, 4659]. These techniques have allowed us to assign all the resonances in the two domains and determine their structure. We found that the individual domains had the same structures as determined by X-ray diffraction in the crystal. However, for the a-helical linker, we didn t find the helical character close to the center of what was supposed to be this helix. Moreover, we found that the rotational diffusion of the protein in the solution did not correspond to a rigid dumbbell shape. The rotational diffusion of each of the two domains was almost isotropic, as if they did not feel each other s presence. This told us that the linker must be extremely flexible [Barbato, G. Ikura, M. Kay, L. E. Pastor, R. W Bax, A. Biochemistry 1992, 3i, 5269]. 

Figure 15 Schematic illustration of three different pairs of triple resonance NMR experiments that can be used for making sequence-specific resonance assignments. Left panel HNCACO and HNCO middle panel HNCA and HN(CO)CA right panel HNCACB and CBCA(CO)NH. In each case, the experiment listed first, which is shown in red, provides intraresidue correlations (and sometimes also interresidue correlations), whereas the experiment listed second, shown in blue, provides only interresidue correlations.

Moseley, H.N.B., Monleon, D., and Montelione, G.T., Automatic Determination of Protein Backbone Resonance Assignments from Triple Resonance NMR Data, Methods in Enzymology, 339, 91, 2001. 

Szyperski T, Wider G, Bushweller JH, Wiithrich K (1993) Reduced dimensionality in triple resonance NMR experiments. J Am Chem Soc 115 9307-9308... 

The nomenclature used to define siloxane compounds combines the use of the letters M, D, T and Q, which represent RsSi—O—R2Si(—O— >2, RSi(—O—)s and Si(—O—)a units respectively, where R stands for aliphatic and/or aromatic substituents or H. In this study, substituents other than methyl groups are indicated as superscripts for example, M = (Me2HSi—O—). Scheme 1 shows the structure of MDsM . We use MD3M , which is a simple but important oligomer of PDMS, to show the use of 3D H/ C7 Si triple resonance NMR for the characterization of PDMS structures. 

The role of peptides in the development of solid-state NMR of aligned samples has been reviewed by Sinha et al In particular, the role of synthetic peptides in the development of triple-resonance methods has been described. Recent developments of pulse sequences and NMR probes for triple-resonance NMR of aligned samples have also been presented. 

The following is a description of a typical run reported in scientific journals with computer-assisted NMR research (attention should be paid to the language of description) NMR experiments are carried out at 35°C on 600, 750, 800, and 900-MHz spectrometers, respectively. Proton, N, and, backbone and side-chain resonances of proteins are assigned by three-dimensional (3D) double- and triple-resonance NMR experiments H resonance for a protein by two-dimensional (2D) C-filtered experiments and P resonance of a protein from a 2D H- P correlation spectmm. 

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