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

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].