A growth hormone agonist produced by targeted mutagenesis at binding site 1: evidence that site 1 regulates bioactivity

Growth hormone (GH) is believed to signal by dimerizing its receptor through two binding sites on the hormone. Previous attempts to increase the biopotency of GH by increasing its site 1 affinity have been unsuccessful, which has led to a bias toward engineering site 2 interactions in the quest for creation of super agonists. Here we report that increasing site 1 affinity can markedly increase proliferative bioactivity in FDC-P1 cells expressing full-length GHR. In contrast, we find three site 1 mutants with affinities for site one similar to or greater than wild type GH, which have markedly decreased bioactivity. Through crystal structure analysis of the receptor interactive regions of these GH analogues, we are able to suggest why previous mutagenesis on human GH failed to improve biopotency, and thus provide a new avenue for GH and cytokine agonist design

Folding and conformational stability of porcine growth hormone.

The large scale production and purification of recombinant-derived porcine growth hormone (pGH) and mutants thereof, bream growth hormone (brGH) and human growth hormone (hGH) was carried out. Each of the protein products were analysed by various physicochemical means, in order to verify their identity and homogeneity. The equilibrium denaturation of pGH using the chemical denaturant guanidine hydrochloride (GuHCl), was monitored by a variety of spectroscopic and hydrodynamic probes. The denaturation of pGH resulted in absorbance- and fluorescence-detected transitions which were coincident, whereas far-UV circular dichroism- and hydrodynamic radius-detected transitions occured at higher concentrations of GuHCl, indicative of a greater stability to denaturation. Conformations intermediate to the folded and fully unfolded states were found to be stable at equilibrium under partially denaturing conditions. These intermediate forms have the characteristics of molten-globule or compact denatured states; a compact structure with considerable helix content, yet possessing a tertiary structure similar to that of the fully unfolded state. At concentrations above 10uM, the intermediate was shown to aggregate, forming a stable associated intermediate. These results suggest that pGH does not follow a simple two-state folding mechanism, but is consistent with the framework model of protein folding. The conformational stabilities of pGH, ten site-directed mutants of pGH, and wild-type bream and human GH, were determined using GuHCl-induced equilibrium denaturation under a standard set of conditions. Single amino acid changes in the sequence of pGH were shown to have different effects on (i) the conformational stability, (ii) the cooperativity of the denaturation transition, i.e., mGuHCl and (iii) the midpoint of the denaturation transition, i.e., [GuHCl] 1/2 . Bream GH was shown to have a stability similar to that of wild-type pGH whereas human GH, in accordance with previously published values [Brems, D. N., Brown, P. L., and Becker, G.W. (1990) J. Biol. Chem. 265, 5504-5511], was found to be significantly more stable than pGH and brGH. One mutant in which a methionine residue was replaced by a tryptophan [pGH(M8)], was found to be significantly more stable than wild-type pGH, due to an increase in both mGuHCl and [GuHCl] 1/2 coincidence of the UV-, fluorescence and hydrodynamic radius-detected equilibrium denaturation curves and the absence of significant amounts of associated forms suggests that pGH(M8) folding/unfolding is more closely approximated by a two-state mechanism than wild-type pGH.Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 199