STRUCTURE AND DYNAMICS OF THE HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR DETERMINED BY NMR-SPECTROSCOPY - LOOP MOBILITY IN A 4-HELIX-BUNDLE PROTEIN

Recombinant N-15- and C-13-labeled human granulocyte colony-stimulatin g factor (rh-metG-CSF) has been studied by 2D and 3D NMR using uniform ly labeled protein, as well as residue-specific N-15-labeled samples. Assignment of 90% of the backbone resonances and 85% of side-chain res onances has enabled the determination of both the secondary and tertia ry structures of the protein. The fold is similar to those of the huma n growth hormone and other growth factors. Four stretches of helices w ere identified between residues 11 and 41 (helix A), 71 and 95 (helix B), 102 and 125 (helix C), and 145 and 170 (helix D), which form a lef t-handed four-helix bundle with helices A and B aligned parallel to on e another (up-up) and antiparallel to helices C and D (down-down). An additional short fifth helix (E) is part of the AB loop connecting hel ices A and B. Examination of the protein's relaxation behavior, based on the model-free approach of Lipari and Szabo, shows that the G-CSF b ackbone has a well-defined structure of limited conformational flexibi lity in helices. In contrast, the long loop connecting helices C and D exhibits substantial fast internal motion compared to the overall rot ational correlation time of the whole molecule, which is on the order of 13 ns.