Staphylococcal nuclease: sequential assignments and solution structure.
Sequential assignments are reported for backbone 15N and 1H of nearly all residues of staphylococcal nuclease (Nase) complexed with thymidine 3',5'-diphosphate and Ca2+. Because of the relatively large size of the Nase ternary complex, Mr 18K, the crucial element of our assignment strategy was the use of isotope-edited two-dimensional NMR spectra, particularly 15N-edited nuclear Overhauser enhancement spectroscopy (NOESY), 15N-edited J-correlated spectroscopy (COSY), and 1H/15N or 1H/13C heteronuclear multiple quantum shift correlation spectroscopy (HMQC). These experiments, together with the more conventional NOESY, COSY, and homonuclear Hartmann-Hahn spectra of natural abundance or deuteriated samples, yielded backbone assignments of 127 of the 136 residues in the structured part of the protein. Using the NOESY data, we identified three helical domains and several beta-sheets which were in close correspondence with secondary structure identified in the crystal structure. Moreover, many long-range NOESY connectivities were identified that were in agreement with distances derived from the crystal structure. The region of the sequence in the neighborhood of residue 50 appears to be more flexible and disordered in solution than in the crystal. Very slowly exchanging amide protons are those found to be hydrogen bonded in the crystal structure; however, even hydrogen-bonded amides located within similar types of regular secondary structures, e.g., alpha-helices, exchange with greatly different rates.
Torchia
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Bone Research Branch, National Institute of Dental Research, Bethesda, Maryland 20892.
Sparks
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Bax
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D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions; D013942:Thymine Nucleotides / Q000378:metabolism
D012996:Solutions; D013942:Thymine Nucleotides; C019111:thymidine 3',5'-diphosphate; D008836:Micrococcal Nuclease; D002118:Calcium