Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease.
This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.
Kay
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Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Torchia
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DA
Bax
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D002118:Calcium / Q000493:pharmacokinetics; D002467:Cell Nucleus / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008836:Micrococcal Nuclease; D009587:Nitrogen Isotopes; D010449:Peptide Mapping; D011487:Protein Conformation; D013816:Thermodynamics; D013942:Thymine Nucleotides
D009587:Nitrogen Isotopes; D013942:Thymine Nucleotides; C019111:thymidine 3',5'-diphosphate; D008836:Micrococcal Nuclease; D002118:Calcium