SECONDARY STRUCTURE AND BACKBONE DYNAMICS OF HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR IN SOLUTION

The secondary structure and backbone dynamics of the cytokine, human g ranulocyte. colony-stimulating factor (hG-CSF) have been determined by heteronuclear nuclear magnetic resonance (NMR) techniques. Virtually complete NH, (CH)-H-alpha, (CH)-H-beta N-15, C-13 alpha, and C-13 beta assignment of the 175-residue recombinant protein, methionyl-[Cys-17- Ser]-hG-CSF, was achieved by use of three-dimensional (3D) heteronucle ar H-1-N-15 and triple-resonance H-1-N-15-C-13 experiments. Spectra re corded at 750 MHz aided the assignment of severely overlapped regions. The structures of G-CSF from several species have recently been deter mined by X-ray diffraction [Hill, C. P., Osslund, T. D., and Eisenberg , D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5167-5171; Lovejoy, B., Cascio, D., and Eisenberg, D. (1993) J. Mol. Biol. 234, 640-653]. Like several cytokines, hG-CSF has a four-helix topology (A-D) with overha nd loop connections, but with an additional helical segment (A') ident ified in the connection between helix A and helix B. The solution-stat e determination of the secondary structure is based on short- and medi um-range NOEs, backbone J-couplings, and NH exchange data and is corro borated by C-l3 alpha secondary shifts. The helices are defined as fol lows: A, 10-38; A', 44-53; B, 71-91; C, 102-123; D, 143-172. The dynam ics of the amide backbone resonances, investigated using H-1-N-15 hete ronuclear NMR, indicate a rigid protein core with some increased mobil ity in the AB loop and more pronounced mobility in the CD loop. Slow c onformational exchange due to proline cis-trans isomerization is sugge sted by satellite resonances observed in two-dimensional heteronuclear spectra for some resonances from residues near prolines.