Recombinant wild-type human IGF-1 and a C-region mutant in which residues 28–37 have been replaced by a 4-glycine bridge (4-Gly IGF-1) were secreted and purified from yeast. An IGF-1 analogue in which residues 29–41 of the C-region have been deleted (mini IGF-1) was created by site-directed mutagenesis and also expressed. All three proteins adopted the insulin-fold as determined by circular dichroism. The significantly raised expression levels of mini IGF-1 allowed the recording of two-dimensional NMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptor was ˜100-fold lower than that of wild-type IGF-1 and the affinity for the insulin receptor was ˜10-fold lower. Mini IGF-1 showed no affinity for either receptor. Not only does the C-region of IGF-1 contribute directly to the free energy of binding to the IGF-1 receptor, but also the absence of flexibility in this region eliminates binding altogether. As postulated for the binding of insulin to its own receptor, it is proposed that binding of IGF-1 to the IGF-1 receptor also involves a conformational change in which the C-terminal B-region residues detach from the body of the molecule to expose the underlying A-region residues.
|Journal||Protein Engineering Design and Selection|
|Publication status||Published - Nov 1996|