Three-dimensional spectroscopy pulse sequences

NOESY NMR spectroscopy is a homonuclear two-dimensional experiment that identifies proton nuclei that are close to each other in space. If one has already identified proton resonances in one-dimensional NMR spectroscopy or by other methods, it is then possible to determine three dimensional structure through NOESY. For instance, it is possible to determine how large molecules such as proteins fold themselves in three-dimensional space using the NOESY technique. The solution structures thus determined can be compared with solid-state information on the same protein obtained from X-ray crystallographic studies. The pulse sequence for a simple NOESY experiment is shown in Figure 3.23 as adapted from Figure 8.12 of reference 19. 

The selection of the pulse sequence to be used out of the hundreds that have been published depends on the information desired. NMR spectroscopy cannot only be used to determine high-quality three-dimensional structures, but can provide information about the global fold, interactions with other molecules or just the identification of the secondary... 

In 2D NMR spectroscopy, a two-dimensional data set is acquired as a function of two time variables tx and t2 as shown schematically in Figure 14.4 [1, 7]. Figure 14.4a shows the general case while the three pulse sequence of Figure 14.4b represents a typical example. 

ID, one-dimensional 2D, two-dimensional 3D, three-dimensional AMP, adenosine monophosphate CNDO, complete neglect of differential overlap COSY, correlation spectroscopy CPMG, Carr-Purcell-Meiboom-Gill NMR pulse sequence CT, constant time dAMP, deoxyadenosine monophosphate DFT, density functional... 

Other pulse sequences are in use such as the three-pulse sequence (Figure 3.16) and hyperfine sublevel correlation (HYSCORE) spectroscopy, the latter being a two-dimensional technique.P ]... 

Three dimensional NMR spectroscopy and improved pulse sequences... 

The assignment of resonances to the chemical structure commences from the main chain atoms HN, N, CA, CO and CB. The experiments, i.e. the pulse sequences for this purpose, appear superficially complex but are in fact built from concatenated parts of heteronuclear polarization transfers. The magnetization is often derived from the amide proton and also from the amide nitrogen when using transverse relaxation optimized spectroscopy. Subsequently the polarization is relayed to amide nitrogen and further to carbons. Three-dimensional spectra are produced by acquiring the frequencies of the amide proton directly and recording... 

Overlap of lines can make analysis difficult when several nuclei contribute in the one-dimensional (ID) two- and three-pulse ESEEM spectra. Eollowing the development in NMR, methods to simplify the analysis involving two-dimensional (2D) techniques have therefore been designed. The Hyperfine Sublevel Correlation Spectroscopy, or HYSCORE method proposed in 1986 [14] is at present the most commonly used 2D ESEEM technique. The HYSCORE experiment has been applied successfully to study single crystals, but is more often applied to orienta-tionally disordered systems. It is a four-pulse experiment (Fig. 2.23(a)) with a k pulse inserted between the second and the third k/2 pulse of the three-pulse stimulated echo sequence. This causes a mixing of the signals due to the two nuclear transitions with m.s = Vi of an 5 = Vi species. For a particular nucleus two lines appear at (v , V ) and (V ", v ) in the 2D spectrum as shown most clearly in the contour map (d) of Fig. 2.23. The lines of a nucleus with a nuclear Zeeman frequency... 

Figure 1. Scheme of the pulse EPR sequences mentioned in this chapter, (a) Two-pulse ESEEM. (b) Three-pulse ESEEM. (c) Four-pulse ESEEM. When times fi and ti are stepped under the constraint of ti= ti= T, combination-peak experiment is performed. Two-dimensional HYSCORE spectroscopy is done using the same sequence, whereby t and are stepped independently. The second and third nil pulse are replaced by high-tuming-angle (HTA) pulses in a matched HYSCORE experiment, (d) SMART-HYSCORE. The first and third pulses are HTA pulses, (e) Davies ENDOR. (f) Mims ENDOR. (g) ELDOR-detected NMR. 

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