1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy.

The assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising of 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation > 95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, C alpha and C beta chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C alpha correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H alpha and H beta protons as well as 3JHNH alpha coupling constants, amide exchange and 13C alpha and 13C beta secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel beta-sheets (residues 30-34, 39-44, 48-53, 62-67, 71-76, 81-85, 91-94, 103-108, 113-118, 123-125 and 148-152) and a helix-like structure (residues 131-136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131-136 were defined as beta-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128-138) instead of the beta-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9-28. This is consistent with the X-ray structures of FGF-2, where the first 17-19 residues were ill defined.