1H,15N,13C and13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

✍ Scribed by Franklin J. Moy; Andrew P. Seddon; Ernest B. Campbell; Peter Böhlen; Robert Powers

• Publisher: Springer Netherlands
• Year: 1995
• Tongue: English
• Weight: 943 KB
• Volume: 6
• Category: Article
• ISSN: 0925-2738
• DOI: 10.1007/bf00197806

✦ Synopsis

The assignments of the ~H, 15N, 13CO and ~3C 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 ~SN-and ~SN-/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 ~, C ~ and H a to the backbone amide ~H and ~SN of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C ~, C ~ and H ~ to the backbone amide ~H and ~SN in the CBCANH and HNHA experiments. In addition, C a and C ~ 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 ~ 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 atiphatic ~H and ~3C 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 ~ and H ~ protons as well as 3JHNHC~ coupling constants, amide exchange and ~3C~ and 13C1~ secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel 13-sheets (residues 30-