¹H, ¹⁵N, ¹³C and ¹³CO Assignments and Secondary Structure Determination of Basic Fibroblast Growth Factor Using 3D Heteronuclear NMR Spectroscopy

Authors

Franklin J. Moy, Department of Structural Biology, Medical Research Division, Wyeth-Ayerst Research, Pearl River, NY
Andrew P. Seddon, Department of Protein Chemistry, Medical Research Division, Wyeth-Ayerst Research, Pearl River, NY
Ernest B. Campbell, Department of Protein Chemistry, Medical Research Division, Wyeth-Ayerst Research, Pearl River, NY
Peter Böhlen, Department of Protein Chemistry, Medical Research Division, Wyeth-Ayerst Research, Pearl River, NY
Robert Powers, University of Nebraska - LincolnFollow

Date of this Version

1995

Citation

Published in Journal of Biomolecular NMR 6:3 (1995), pp. 245–254.

Comments

Copyright © 1995 ESCOM Science Publishers B.V. Used by permission.

Abstract

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 cor-relation of C^α, C^β, and H^α to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experi-ments. 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 HNCA experiment. Aliphatic side-chain spin sys-tems 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, Hα, and Hβ protons as well as ³J_H^N_H^α coupling constants, amide exchange, and 13Cα and 13Cβ 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.