Spectroscopy large molecules

The advantages of resonance Raman spectroscopy have already been discussed in section BL2.2.3. For these reasons it is rapidly becoming the method of choice for studying large molecules in solution. Flere we will present one study that exemplifies its attributes. There are two complementary methods for studying proteins. 

NOE-difFerence spectroscopy is particularly valuable for distinguishing stereoisomers, for it relies solely on intemuclear distances, and thus avoids any problems of ambiguity or absence associated with couplings. With smallish molecules, it is best carried out in the above 1D maimer, because 2 s are necessary for tire transmission of the NOE. The transmission process becomes more efficient with large molecules and is almost optimal for proteins. However, problems can occur with molecules of intemiediate size [3f]. A 2D version of the NOE-difference experiment exists, called NOESY. 

For large molecules, such as proteins, the main method in use is a 2D technique, called NOESY (nuclear Overhauser effect spectroscopy). The basic experiment [33, 34] consists of tluee 90° pulses. The first pulse converts die longitudinal magnetizations for all protons, present at equilibrium, into transverse magnetizations which evolve diirhig the subsequent evolution time In this way, the transverse magnetization components for different protons become labelled by their resonance frequencies. The second 90° pulse rotates the magnetizations to the -z-direction. 

The next most useful is vibrational spectroscopy but identification of large molecules is still uncertain. In the laboratory, vibrational spectroscopy in the infrared (IR) is used routinely to identify the functional groups in organic molecules but although this is important information it is not sufficient to identify the molecule. Even in the fingerprint region where the low wavenumber floppy vibrational modes of big molecules are observed, this is hardly diagnostic of structure. On occasion, however, when the vibrational transition can be resolved rotationally then the analysis of the spectrum becomes more certain. 

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. 

Doppler-Free Time-Resolved Polarization Spectroscopy of Large Molecules Measurement of Excited State Rotational Constants, J. S. Baskin, P. M. Felker, and A. H. Zewail, J. Chem. Phys. 84, 4708 (1986). 

The development of a new form of spectroscopy based on the exploitation of the time evolution of the coherence associated with the rotational motion of an excited molecule. Conventional spectroscopies depend on the measurement of differences between the energy levels of a molecule, which become more and more difficult to measure and to interpret as the size of the molecule increases. In contrast, the intervals between recurrences in the coherent rotational motions of large molecules are directly related to the moments of inertia of the molecules and can be used to determine their structures. 

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