Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal. growth factor, [Abu6, 20] mEGF 4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48 , have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was desig ned to determine the contribution of the 6-20 disulfide bridge to the struc ture and function of mEGF The overall structure of this analogue was simila r to that of native mEGF, indicating that the loss of the 6-20 disulfide br idge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta -sheet from their position in native mEGF Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant lo ss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A 431 cells compared with native mEGF and mEGF4-48, implying that the structu ral changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, wer e sufficient to interfere with receptor binding. The loss of binding affini ty probably arose mainly from steric interactions of the dislocated N-termi nal region with part of the receptor binding surface of EGF [G1, M3, K21, H 40] mEGF1-48 was also synthesized in order to compare the synthetic polypep tide with the corresponding product of recombinant expression. Its mitogeni c activity in Balb/c 3T3 cells was similar to that of native mEGF and analy sis of its H-1 chemical shifts suggested that its structure was also very s imilar to native.