Assignments of 1H-15N magnetic resonances and identification of secondary structure elements of the lambda-cro repressor

Abstract

The assignments of 1H-15N magnetic resonances of the lambda-cro repressor are presented. Individual 15N-amino acids were incorporated into the protein, or it was uniformly labeled with 15N. For the 13C-15N double-labeling experiments, 13C-amino acids were incorporated into the uniformly 15N-labeled protein. All the amide 1H-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 2D 1H-1H correlation spectroscopies were applied to the assignment of the side-chain protons. Some of the 1H 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 alpha-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 beta-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].