Multidimensional NMR experiments

The centre band RF field of a PIP is usually used to achieve a frequency-shifted excitation while preserving the phase coherence among the RF pulses applied before and after the PIP. The phase coherence in RF pulses is crucial in some of the multidimensional NMR experiments, especially in the 13C dimension where the 13CO and 13C need to be excited separately. Unpredictable results may occur if the phase coherence in RF pulses fails in some NMR experiments. 

Although the first NMR spectra of deuterated proteins were published already in 1968, the deuteration was mainly employed for the purpose of simplifying one-dimensional proton spectra.53,54 Later in the 1980s, LeMaster and co-workers employed random fractional deuteration for the assignment of thioredoxin using homonuclear two-dimensional spectroscopy.55 However, it was realized that deuteration significantly improves the spectral quality of homonuclear multidimensional NMR experiments. 

Limited protein stability often hampers successful structure elucidation by X-ray crystallography and/or NMR spectroscopy. Relaxation properties are usually improved at elevated temperatures, and multidimensional NMR experiments require sample lifetimes to extend over several days to weeks in order to acquire all the necessary data. In addition, the activity of contaminating proteases that are sometimes present in purified samples can be significant at the experimental temperatures. Therefore, the stability of a target protein can be a concern, in particular for expensive isotope-labeled proteins. 

NMR is a remarkably flexible technique that can be effectively used to address many analytical issues in the development of biopharmaceutical products. Although it is already more than 50 years old, NMR is still underutilized in the biopharmaceutical industry for solving process-related analytical problems. In this chapter, we have described many simple and useful NMR applications for biopharmaceutical process development and validation. In particular, quantitative NMR analysis is perhaps the most important application. It is suitable for quantitating small organic molecules with a detection limit of 1 to 10 p.g/ml. In general, only simple one-dimensional NMR experiments are required for quantitative analysis. The other important application of NMR in biopharmaceutical development is the structural characterization of molecules that are product related (e.g., carbohydrates and peptide fragments) or process related (e.g., impurities and buffer components). However, structural studies typically require sophisticated multidimensional NMR experiments. 

Over the last 10-15 years, multidimensional (3D and 4D) NMR spectroscopic and computational techniques have been developed to solve structures of larger proteins in solution. This technology has the advantage of resolving the severe overlap in 2D NMR spectra for proteins having >100 residues. Usually, multidimensional NMR experiments require isotopic labeling of some or all nuclei of interest. For example, signal overlap in a two-dimensional correlation spectrum can be resolved in the dimension of the heteronucleus—that is, N or attached to the protons of relevance. If fast relaxation is a problem,... 

The advances in NMR spectroscopy in the last ten years were enormous. Thus, almost all laboratories which produce papers are equipped with SCM (Super Conducting Magnet) instruments with 400-500 MHz magnets, and 2D or multidimensional NMR experiments are now routinely employed. In addition, solid-state NMR and NMR imaging (MRI) have widened their scope to a considerable extent. 29 Si NMR has enjoyed this general progress. 

High resolution multidimensional NMR experiments can provide the dendrimer chemist with a wealth of additional information extending far beyond the determination of the molecular structure. In the interpretation of (2D)-NOESY (NOESY=nuclear Overhauser enhancement spectroscopy) spectra, a knowledge of the spatial interrelationships between protons in different parts of the dendrimer scaffold can be acquired from proton-proton NOE interactions. At the same time, the prevailing conformation of the dendritic branches in the solvent used can be deduced from this information. Furthermore, studies of dendrimer/sol-vent interactions and the influence of solvent on the spatial structure of the dendrimer are also possible [22]. Thus the information content of such NMR experiments resembles that of small-angle scattering experiments on dissolved dendrimers (see Section 7.6). 

Yang, D. W., Konrat, R., and Kay, L. E. (1997). A multidimensional NMR experiment for measurement of the protein dihedral angle psi based on cross-correlated relaxation between (H alpha-13C alpha) XH dipolar and 13C (carbonyl) chemical shift anisotropy mechanisms. J. Am. Chem. Soc. 119,11938-11940. 

A. Perczel, A.G. Csaszar, Toward direct determination of conformations of protein building units from multidimensional NMR experiments I. A theoretical case study of For-Gly-NH2 and For-L-Ala-NH2. J. Comput. Chem. 21, 882-900 (2000)... 

In Chapter 11 we shall also introduce the product operator formalism, in which the basic ideas of the density matrix are expressed in a simpler algebraic form that resembles the spin operators characteristic of the steady-state quantum mechanical approach. Although there are some limitations in this method, it is the general approach used to describe modern multidimensional NMR experiments. 

As we have seen in the examples in Section 10.2, most 2D experiments involve transfer of polarization or coherence, sometimes in multistep processes in which the efficiency of each is far less than 100%. Thus, great care must be used in designing such experiments to craft each step carefully to optimize the final signal. Also, extensive phase cycling that is required in some multidimensional NMR experiments extends the minimum time required for the study. If signals are weak and extensive time averaging is needed anyway to obtain adequate... 

Forgo, P., Hohmann, J., Dombi, G., Mathe, L. (2004) Advanced Multidimensional NMR Experiments as Tools for Structure Determination of Amaryllidaceae Alkaloids, In Acamovic, T. Steward, S. Pennycott T. W. (Eds.) Poisonous Plants and Related Toxins, CAB International, Wallingford, UK, 322-328. 

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 multidimensional NMR experiments that contain several evolution and mixing periods, even more combinations are possible (Griesinger et al., 1987b). In these experiments, Hartmann-Hahn mixing periods with in-phase coherence transfer are of particular advantage, because the resolution is often limited in the indirectly detected frequency dimensions. 

Exactly the same results were obtained with spin diffusion experiments performed at ZSM-39. Frequencies can be affected by spin diffusion between sites having different NMR parameters, when, for example, magnetization is transported through a solid by means of mutual spin "flip-flops" that can occur even in the absence of atomic or molecular motion. By monitoring the correlation among frequencies in the different dimensions of a multidimensional NMR experiment, it is possible to learn about the mechanisms and rates of reorientation and diffusion processes in solids (32). 

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. 

Several strategies have been developed to measure residual dipolar couplings in 13C-,15N-labelled proteins. Residual dipolar couplings can be measured in the frequency domain of multidimensional spectra of 13C- or 15N-labelled proteins by not decoupling in either the direct or indirect dimension.107154 Alternatively, residual dipolar couplings can be measured from the J-modulation of the cross-peak intensity in multidimensional NMR experiments.155156 Frequency-based experiments are the simplest to implement but sometimes suffer because of the complexity of the spectrum created by twice the number of cross-peaks. This latter drawback may be overcome by simple editing techniques.154-157-159 Intensity-based experiments are more prone to... 

Multidimensional NMR experiments also provide additional information that is unavailable from onedimensional (ID) spectra even in the limit of high-resolution. For example, 2D 2H exchange experiments, which identify a particular change in molecular orientation between two evolution periods, have already been mentioned above.9 For dipolar-coupled nuclei, the combination of fast MAS with 2D multiple-quantum (MQ) spectroscopy achieves high resolution, while allowing the structural and dynamic information inherent to the dipolar couplings to be accessed. Specifically, using the homonuclear H- H double-... 

The evolution of the many-molecule dynamics, with more and more units participating in the motion with increasing time, is mirrored directly in colloidal suspensions of particles using confocal microscopy [213]. The correlation function of the dynamically heterogeneous a-relaxation is stretched over more decades of time than the linear exponential Debye relaxation function as a consequence of the intermolecularly cooperative dynamics. Other multidimensional NMR experiments [226] have shown that molecular reorientation in the heterogeneous a-relaxation occurs by relatively small jump angles, conceptually simlar to the primitive relaxation or as found experimentally for the JG relaxation [227]. 

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