Assignments of1H-15N magnetic resonances and identification of secondary structure elements of the λ-cro repressor

H. Matsuo, M. Shirakawa, T. Ohkubo, T. Yamazaki, Y. Kyogoku

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Abstract

The assignments of1H-15N magnetic resonances of the λ-cro repressor are presented. Individual15N-amino acids were incorporated into the protein, or it was uniformly labeled with15N. For the13C-15N double-labeling experiments,13C-amino acids were incorporated into the uniformly15N-labeled protein. All the amide1H-15N resonances could be assigned with such specific labeling, and sequential connectivities obtained by two-dimensional (2D)1H-15N reverse correlation spectroscopies and three-dimensional (3D)1H/15N NOESY-HMQC spectroscopy. Conventional 2D1H-1H correlation spectroscopies were applied to the assignment of the side-chain protons. Some of the1H resonance assignments are inconsistent with those previously reported [Weber, P.L., Wemmer, D.E. and Reid, B.R. (1985)Biochemistry, 24, 4553-4562]. The sequential NOE connectivities and H-D exchange rates indicate several elements of the secondary structure, including α-helices consisting of residues 8-15, 19-25 and 28-37, and three extended strands consisting of residues 4-7, 39-45 and 49-55. Based on several long-range NOEs, the three extended strands could be combined to form an antiparallel β-sheet. The amide proton resonances of the C-terminal residues except Ala66 (residues 60-65) were hardly observed at neutral pH, indicating that the arm is flexible. The identified secondary structure elements in solution show good agreement with those in the crystal structure of the cro protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y. and Matthews, B.W. (1981)Nature, 290, 754-758].

Original languageEnglish (US)
Pages (from-to)191-204
Number of pages14
JournalJournal of biomolecular NMR
Volume1
Issue number2
DOIs
StatePublished - Jul 1991

Keywords

  • 3D NMR
  • Assignment: λ-cro
  • HMQC
  • Isotope labeling
  • SQC
  • Secondary structure

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