Correlated spectroscopy data requirements

Scalar coupled experiments COSY and TOCSY The correlated spectroscopy (COSY) experiment is one of the most simple 2D-NMR pulse sequences in terms of the number of RF pulses it requires [32]. The basic sequence consists of a 90-C-90-acquire. The sequence starts with an excitation pulse followed by an evolution period and then an additional 90° pulse prior to acquisition. Once the time domain data are Fourier transformed, the data appear as a diagonal in... 

New techniques for data analysis and improvements in instrumentation have now made it possible to carry out stmctural and conformational studies of biopolymers including proteins, polysaccharides, and nucleic acids. NMR, which may be done on noncrystalline materials in solution, provides a technique complementary to X-ray diffraction, which requires crystals for analysis. One-dimensional NMR, as described to this point, can offer structural data for smaller molecules. But proteins and other biopolymers with large numbers of protons will yield a very crowded spectrum with many overlapping lines. In multidimensional NMR (2-D, 3-D, 4-D), peaks are spread out through two or more axes to improve resolution. The techniques of correlation spectroscopy (COSY), nuclear Overhausser effect spectroscopy (NOESY), and transverse relaxation-optimized spectroscopy (TROSY) depend on the observation that nonequivalent protons interact with each other. By using multiple-pulse techniques, it is possible to perturb one nucleus and observe the effect on the spin states of other nuclei. The availability of powerful computers and Fourier transform (FT) calculations makes it possible to elucidate structures of proteins up to 40,000 daltons in molecular mass and there is future promise for studies on proteins over 100,000... 

Polarization Fluctuation Spectroscopy is a relatively simple extension to the well established technique of Photon Correlation Spectroscopy. The optical equipment required in addition to that used for PCS is standard, as also is the data processing hardware for determining the correlation functions. The means to analyze the data is now achievable because of the computational techiuques for calculating the scattering properties for particles of complex shape and composition that have been developed in the last decade. The additional functionality afforded by PFS is achieved at modest additional cost, and importantly, by performing measurements that are essentially the same as those for PCS. 

In the Dynamic Light Scattering method (also known as Photon Correlation Spectroscopy), one measures the diffusion of macromolecular chains in solution. The diffusion coefficient is related to the molar mass. The method suffers a number of difficulties. Raw experimental data do not lend themselves to immediate interpretation and must be fitted using a model MMD. Furthermore, D5mamic LS cannot be used to choose between a unimodal MMD and bimodal MMD. In fact data are fitted using a model MMD and the number of parameters to be fitted increases quickly, going from unimodal to bimodal (a bimodal MMD requires finding the best fit of six parameters). 

I can thus conclude by recalling what I said at the beginning of my talk, namely that the future of these studies, if any, lies in the possibility of developing more and more refined experimental techniques able to give a detailed description of the sequence distribution in copolymers and at present only NMR and to a minor extent IR spectroscopy fulfil these requirements then I hope that the new concepts of copolymer statistics discussed here could represent, for the scientists dealing with structural analysis of chemical and steric copolymers by NMR, a wider framework for representing and correlating their data. 

The low MW power levels conuuonly employed in TREPR spectroscopy do not require any precautions to avoid detector overload and, therefore, the fiill time development of the transient magnetization is obtained undiminished by any MW detection deadtime. (3) Standard CW EPR equipment can be used for TREPR requiring only moderate efforts to adapt the MW detection part of the spectrometer for the observation of the transient response to a pulsed light excitation with high time resolution. (4) TREPR spectroscopy proved to be a suitable teclmique for observing a variety of spin coherence phenomena, such as transient nutations [16], quantum beats [17] and nuclear modulations [18], that have been usefi.il to interpret EPR data on light-mduced spm-correlated radical pairs. 

When performing 2D-NMR experiments one must keep in mind that the second frequency dimension (Fx) is digitized by the number of tx increments. Therefore, it is important to consider the amount of spectral resolution that is needed to resolve the correlations of interest. In the first dimension (F2), the resolution is independent of time relative to F. The only requirement for F2 is that the necessary number of scans is obtained to allow appropriate signal averaging to obtain the desired S/N. These two parameters, the number of scans acquired per tx increment and the total number of tx increments, are what dictate the amount of time required to acquire the full 2D-data matrix. 2D-homo-nuclear spectroscopy can be summarized by three different interactions, namely scalar coupling, dipolar coupling and exchange processes. 

Such ambiguity and also the low structural resolution of the method require that the spectroscopic properties of protein fluorophores and their reactions in electronic excited states be thoroughly studied and characterized in simple model systems. Furthermore, the reliability of the results should increase with the inclusion of this additional information into the analysis and with the comparison of the complementary data. Recently, there has been a tendency not only to study certain fluorescence parameters and to establish their correlation with protein dynamics but also to analyze them jointly, to treat the spectroscopic data multiparametrically, and to construct self-consistent models of the dynamic process which take into account these data as a whole. Fluorescence spectroscopy gives a researcher ample opportunities to combine different parameters determined experimentally and to study their interrelationships (Figure 2.1). This opportunity should be exploited to the fullest. 

Raman spectroscopy s sensitivity to the local molecular enviromnent means that it can be correlated to other material properties besides concentration, such as polymorph form, particle size, or polymer crystallinity. This is a powerful advantage, but it can complicate the development and interpretation of calibration models. For example, if a model is built to predict composition, it can appear to fail if the sample particle size distribution does not match what was used in the calibration set. Some models that appear to fail in the field may actually reflect a change in some aspect of the sample that was not sufficiently varied or represented in the calibration set. It is important to identify any differences between laboratory and plant conditions and perform a series of experiments to test the impact of those factors on the spectra and thus the field robustness of any models. This applies not only to physical parameters like flow rate, turbulence, particulates, temperature, crystal size and shape, and pressure, but also to the presence and concentration of minor constituents and expected contaminants. The significance of some of these parameters may be related to the volume of material probed, so factors that are significant in a microspectroscopy mode may not be when using a WAl probe or transmission mode. Regardless, the large calibration data sets required to address these variables can be burdensome. 

Symmetry relationship of cross peak locations has been used for improving the quality of correlation spectra for quite a long time in NMR spectroscopy both for diagonally [21] and laterally symmetric spectra [22]. Such data processing procedures have their advantage but may introduce artifacts and remove real information and therefore should be used with caution [10]. 2Q-HoMQC spectra can be symmetrized directly using the appropriate symmetry function [33], but most commercial software do not provide such option. Also, fine structure of the direct correlation peaks in 2Q-H0MQC spectra is antisymmetric in the SQ dimension which requires extra attention. 

Two conformations of EpoA in complex with tubulin have been proposed on the basis of EC [26] and NMR [76, 96] data, respectively (Fig. 11). The tubulin-bound conformation of EpoA was determined by solution NMR spectroscopy [96] before the EC structure of EpoA bound to tubulin was available. The observation that, in a 100 1 mixture with tubulin, NOE cross-peaks of EpoA have negative sign, indicated that there is a fast exchange equilibrium in solution. This offered the opportunity to measure transferred NMR experiments, that report on the bound conformation of the ligand. A total of 46 interproton distances were derived from cross-peak volumes in tr-NOE spectra. However, these distance restraints did not suffice to define a unique conformation, as several distinct structures were consistent with them. Transferred cross-correlated relaxation (Sect. 2.2.1.3) provided the additional dihedral restraints that were crucial to define the bound conformation [96, 97], One requirement to measure CH-CH dipolar and CH-CO dipolar-CSA CCR rates is that the carbon atoms involved in the interaction are labeled with 13C. The availability of a 13C-labeled sample of EpoA offered the opportunity to derive seven of these dihedral angle restraints from tr-CCR measurements (Fig. 12). 

In the case of an unknown chemical, or where resonance overlap occurs, it may be necessary to call upon the full arsenal of NMR methods. To confirm a heteronuclear coupling, the normal H NMR spectrum is compared with 1H 19F and/or XH 31 P NMR spectra. After this, and, in particular, where a strong background is present, the various 2-D NMR spectra are recorded. Homonuclear chemical shift correlation experiments such as COSY and TOCSY (or some of their variants) provide information on coupled protons, even networks of protons (1), while the inverse detected heteronuclear correlation experiments such as HMQC and HMQC/TOCSY provide similar information but only for protons coupling to heteronuclei, for example, the pairs 1H-31P and - C. Although interpretation of these data provides abundant information on the molecular structure, the results obtained with other analytical or spectrometric techniques must be taken into account as well. The various methods of MS and gas chromatography/Fourier transform infrared (GC/FTIR) spectroscopy supply complementary information to fully resolve or confirm the structure. Unambiguous identification of an unknown chemical requires consistent results from all spectrometric techniques employed. 

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