Methods in Enzymology Vol.227 Metallobiochemistry, Part D: Physical and Spectroscopic Methods

Although the presence of paramagnetic centers in the active site of proteins may severely limit the use of NMR for structural studies, it may also contribute to important advantages in studying the physicochemical properties of the paramagnetic center, as well as the structural arrangement around it. The increased resolution resulting from hyperfine shifts makes it possible to observe the resonances of nuclei close to the active site. The strong and precise geometrical dependence of the paramagnetic contribution to the chemical shift and relaxation of these nuclei can give important information on the local architecture of the active site even in the absence of a global structure determination. The sensitivity of the paramagnetic perturbations makes it an extremely useful technique to probe even small structural changes in the protein active site caused by modifications of physicochemical properties, solution parameters, such as pH, temperature, and ionic strength, as well as binding to substrates, inhibitors, or other proteins. In particular, the dispersion of 13C resonances adds a new and sensitive probe for very small changes. Another useful and relatively fast application is for comparison of the active site architecture in solution for homologous native proteins or proteins subjected to site-specific mutagenesis. 2D NMR can be used in essentially all the cases for which 1D NMR has proved to be an extremely useful structural technique. The use of 2D experiments to study the immediate environment of the active site has obvious advantages over the 1D analogs for cases where the resonances are within the crowded regions.

• Series: Methods in Enzymology (Book 227)
• Hardcover: 634 pages
• Publisher: Academic Press; 1 edition (October 20, 1993)
• Language: English
• ISBN-10: 0121821285
• ISBN-13: 978-0121821289
• Product Dimensions: 6 x 1.4 x 9 inches

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