Structure-function studies of the human interferon gamma

The present study involves the investigation of the structure-function relationships of the human interferon gamma (HuIFN-{dollar}gamma{dollar}). This study is divided into two parts. In the first part, deletion variants were either isolated as a proteolytic cleavage product or constructed by specific gene modifications. Two of the variant polypeptides which had, respectively, 15 and 21 amino acid residues deleted from the C-terminus were successfully expressed in Escherichia coli and were purified by immunoaffinity chromatography to homogeneity. These two variant polypeptides were shown to have specific activities comparable to the parental HuIFN-{dollar}gamma{dollar} molecule. Thus, the functional site(s) of the HuIFN-{dollar}gamma{dollar} must be located in the N-terminal 122 amino acid residues. The variant which had 21 residues deleted was treated with guanidine hydrochloride and was subsequently renatured in vitro. It was found that the antiviral specific activity of the renatured molecule was significantly reduced. Deletion variant polypeptides which had, respectively, 26, 32, and 37 amino acid residues deleted from the C-terminus exhibited reduced intracellular accumulation and attempts to purify these polypeptides were unsuccessful. The biological activities of these latter variants were undetectable at the limit of sensitivity of the antiviral assay. Thus, deletions of more than 21 residues from the C-terminus might have caused structural instability of the molecules which renders these molecules susceptible to proteolytic degradation inside the bacterial cell.; In the second part, single-site variants were constructed by site-directed mutagenesis. The first variant, which was constructed by the substitution of Asp{dollar}sp{lcub}41{rcub}{dollar} for Ala, was found to have significantly reduced antiviral specific activity as well as diminished intracellular accumulation. Secondary structure analysis showed that a conserved {dollar}beta{dollar}-turn was disrupted as a result of this substitution. Since this {dollar}beta{dollar}-turn is highly exposed and is conserved among IFNs-{dollar}gamma{dollar} from various vertebrate species, it is likely to play an important role in intermolecular interactions. A second variant was constructed by replacing Asp{dollar}sp{lcub}41{rcub}{dollar} with Asn. This variant retained the conserved {dollar}beta{dollar}-turn structure; however, the local hydrophilicity was reduced and the negative charge on the carboxyl group of Asp{dollar}sp{lcub}41{rcub}{dollar} was abolished. This purified variant polypeptide was found to have antiviral specific activity comparable to the unmodified molecule. Two variants were also constructed to study the possible influence of the adjacent secondary structures on the conserved {dollar}beta{dollar}-turn. Two residues, Gln{dollar}sp{lcub}46{rcub}{dollar} and Gln{dollar}sp{lcub}48{rcub}{dollar}, were individually replaced by Leu. Hydrophilicity and secondary structure analyses showed that these two substitutions resulted in the reduction of the local hydrophilicity without any alteration in the local secondary structures. Unexpectedly, these purified variant polypeptides were found to have approximately 30-fold increase in antiviral specific activity. (Abstract shortened by UMI.)...