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 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^α, C^β and H^α 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^α, C^β and H^α to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C^α and C^β 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^α correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C^α 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^α and H^β protons as well as ³J_Hn_Hα coupling constants, amide exchange and ¹³C^α and ¹³C^β secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel β-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 β-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128-138) instead of the β-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.