Use of the carbonyl chemical shift to relieve degeneracies in triple-resonance assignment experiments.
We illustrate an approach that uses the backbone carbonyl chemical shift to relieve resonance overlaps in triple-resonance assignment experiments conducted on protein samples. We apply this approach to two cases of simultaneous overlaps: those of ((1)H(N), (15)N) spin pairs and those of ((1)H(alpha), (13)C(alpha)) spin pairs in residues preceding prolines. For these cases we employed respectively CBCACO(N)H and H(CA)CON experiments, simple variants of the commonly used CBCA(CO)NH and HCA(CO)N experiments obtained by replacing one of the indirect dimensions with a carbonyl dimension. We present data collected on ribosomal protein S4 using these experiments, along with overlap statistics for four other polypeptides ranging in size from 76 to 263 residues. These data indicate that the CBCACO(N)H, in combination with the CBCA(CO)NH, can relieve >83% of the ((1)H(N), (15)N) and ((1)H(N), (13)C') overlaps for these proteins. The data also reveal how the H(CA)CON experiment successfully completed the assignment of triply and quadruply degenerate X-Pro spin systems in a mobile, proline-rich region of S4, even when X was a glycine. Finally, we discuss the relative sensitivities of these experiments compared to those of existing sequences, an analysis that reinforces the usefulness of these experiments in assigning extensively overlapped and/or proline-rich sequences in proteins.
Sayers
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Molecular Structural Biology Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, MSC 4307, Bethesda, Maryland 20892-4307, USA.
Torchia
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D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011392:Proline / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D012269:Ribosomal Proteins; C031798:ribosomal protein S4; D011392:Proline