Secondary structure and backbone dynamics of human granulocyte colony-stimulating factor in solution.
The secondary structure and backbone dynamics of the cytokine, human granulocyte colony-stimulating factor (hG-CSF) have been determined by heteronuclear nuclear magnetic resonance (NMR) techniques. Virtually complete NH, C alpha H, C beta H 15N, 13C alpha, and 13C beta assignment of the 175-residue recombinant protein, methionyl-[Cys-17-Ser]-hG-CSF, was achieved by use of three-dimensional (3D) heteronuclear 1H-15N and triple-resonance 1H-15N-13C experiments. Spectra recorded at 750 MHz aided the assignment of severely overlapped regions. The structures of G-CSF from several species have recently been determined by X-ray diffraction [Hill, C. P., Osslund, T. D., & Eisenberg, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5167-5171; Lovejoy, B., Cascio, D., & Eisenberg, D. (1993) J. Mol. Biol. 234, 640-653]. Like several cytokines, hG-CSF has a four-helix topology (A-D) with overhand loop connections, but with an additional helical segment (A') identified in the connection between helix A and helix B. The solution-state determination of the secondary structure is based on short- and medium-range NOEs, backbone J-couplings, and NH exchange data and is corroborated by 13C alpha secondary shifts. The helices are defined as follows: A, 10-38; A',44-53; B, 71-91; C, 102-123; D, 143-172. The dynamics of the amide backbone resonances, investigated using 1H-15N heteronuclear NMR, indicate a rigid protein core with some increased mobility in the AB loop and more pronounced mobility in the CD loop.(ABSTRACT TRUNCATED AT 250 WORDS)
Werner
J M
JM
Department of Biochemistry, University of Oxford, U.K.
Breeze
A L
AL
Kara
B
B
Rosenbrock
G
G
Boyd
J
J
Soffe
N
N
Campbell
I D
ID
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012996:Solutions
D011994:Recombinant Proteins; D012996:Solutions; D016179:Granulocyte Colony-Stimulating Factor