We have previously described a method for producing recombinant methionyl bovine PRL (Met-bPRL), which is as bioactive as the authentic hormone in the Nb2 cell lactogen bioassay; in contrast, a Met-bPRL variant lacking tyrosine 28 was essentially devoid of bioactivity. In the present study we have investigated this loss of bioactivity at the molecular level by determining the bioactivities of a number of Met-bPRL variants engineered to contain specific changes in their primary structures. It was found that the presence of tyrosine per se at the 28 position in Met-bPRL was not essential for high bioactivity, since Met-bPRL variants prepared by replacing tyrosine 28 with other amino acids (arginine, phenylalanine, alanine, and histidine) still had substantial bioactivity (40-74% that of Met-bPRL). Neither was the loss of bioactivity related to a shift in the relative positions of conserved histidines 27 and 30; in fact, histidine 27 was found not to be essential for the bioactivity of the hormone. The loss of bioactivity after deletion of tyrosine 28 from Met-bPRL appears to be related to the removal of an amino acid from the middle of a putative helix (no. 1) rather than to the loss of a residue specific to lactogen function. This suggestion is supported by the finding that Met-bPRL variants obtained by deletion of selected single amino acids from center domains of putative helix 2, 3, or 4 were also essentially devoid of bioactivity. It is speculated that this lack of bioactivity reflects an inability of the proteins to assume a native conformation.
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Luck et al. (1990) studied this question.