H-1, N-15, C-13, AND (CO)-C-13 ASSIGNMENTS AND SECONDARY STRUCTURE DETERMINATION OF BASIC FIBROBLAST GROWTH-FACTOR USING 3D HETERONUCLEAR NMR-SPECTROSCOPY

The assignments of the H-1, N-15, (CO)-C-13 and C-13 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 N-15- and N-15-/C-13-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 H-1 and N-15 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 H-1 and N-15 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. Aliphalic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic H-1 and C-13 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 (3)J(H)N(H) alpha coupling constants, amide exchange and C-13(alpha) and C-13(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 light 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 Xray structures of FGF-2, where the first 17-19 residues were ill defined.