Nuclear Magnetic Resonance Spectroscopy

The nuclear magnetic resonance (NMR) method for studying protein conformation is based on the fact that "nuclei of the same element in different chemical environments give rise to distinct chemically shifted spectral lines." The use of chemical shifts for the identification of specific secondary structures, however, has not been successful in determining secondary structures other than a-helix. The introduction of the nuclear Overhauser effect (NOE) into NMR has provided a network of short, intramolecular distance constraints between distinct locations along the polypeptide chain, which has allowed the characterization of other secondary structures. NOE is the enhancement in amplitude of the spectral line of nucleus observed during irradiation of one of a pair of coupled nuclear spins; irradiation of nuclei can cause changes in the population of the spin-state of the nuclei. Proton-proton NOE distance information was used for identification of polypeptide secondary structures in noncrystalline proteins. The combined information on all of the distances obtained from visual inspection of the two-dimensional NOE (NOESY) spectra was sufficient for determination of helical and /?-sheet secondary structures in small globular proteins.

Although for larger proteins magnetic relaxation becomes a limiting factor, the benefits of using uniform highlevel (>96%) deuteration was shown to inhibit relaxation processes (27). Complete deuteration provides significant signal-to-noise enhancement in heteronuclear NMR assignment and structure determination experiments, which uses the amide proton for detection. NMR pulse sequences lose sensitivity as the size of the protein under study increases above 25 kDa, mainly because of fast 13C transverse relaxation via the strong dipolar coupling between a 13C nucleus and its directly bonded protons. Since the gyro-magnetic ratio of 2H is 6.5 times smaller than that of *H, per deuteration dramatically reduces this relaxation.

A recent news release stated that "A technique called TROST (transverse relaxation-optimized spectroscopy) now greatly reduces NMR line broadening with increasing molecular size, permitting NMR analysis of molecules far beyond 100 kDa in size" (28).

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