RECEPTOR-BINDING AFFINITIES OF HUMAN EPIDERMAL GROWTH-FACTOR VARIANTSHAVING UNNATURAL AMINO-ACID-RESIDUES IN POSITION-23

Ile-23 of human epidermal growth factor (hEGF) has been indicated, by mutagenesis and NMR studies, to be directly recognized by the receptor . In the present study, an unnatural phenylalanine analog, either 2-az aphenylalanine (2aF), 3-azaphenylalanine (3aF), 4-azaphenylalanine (4a F), or 4-fluorophenylalanine (4fF), was incorporated by an in vivo pro tein synthesis system into position 23 of [Phe(23)] hEGF, which retain s appreciable receptor-binding affinity (about 20% of the wild type). We compared the receptor-binding affinities of the variants with that of [Phe(23)]hEGF and found that substitution of Phe-23 with 2aF or 3aF raised the affinity, while substitution with 4aF or 4fF remarkably re duced the affinity. The tertiary structure of [Phe(23)] hEGF was not s ignificantly affected by the substitution of Phe-23 with 2aF, as shown by the two-dimensional nuclear magnetic resonance analysis. In additi on, the substitution of residue 23 with His or Tyr produced an hEGF va riant with a slightly higher receptor-binding affinity than that of [P he(23)]hEGF. Our results suggest that the receptor has an asymmetric h ydrophobic pocket for recognition of the side chain in position 23 of hEGF. Furthermore, on the receptor surface, this pocket seems to be ad jacent to a less hydrophobic region with a hydrogen-bond acceptor and donor. Thus, the use of unnatural amino acids in addition to the 20 na tural ones allows analyses of the structure-function relationship of a protein at a higher resolution than conventional site-directed substi tution by only natural amino acid residues.