Conformational analysis and proteolytic processing of synthetic pre-pro-GnRH/GAP protein

Homogeneous pre-pro-GnRH/GAP protein was recently synthesized in 100 mg quantities by solid-phase methods and surprisingly, the synthetic pre-pro-protein, which normally does not escape the endoplasmic recticulum, was found to inhibit the release of prolactin from cultured pituitary cells. This is the first demonstration of significant biological activity associated with a precursor protein and provides the rationale for its further study. We now report the results of our initial examination of the conformational properties of pre-pro-GnRH/GAP protein as a prelude to solving its solution phase conformation by homonuclear1H-NMR protocols. Thermal andpH titration fluorescence and circular dichroism spectroscopies reveal that the protein is resistant to thermal-induced conformational changes but is particularly sensitive topH-induced conformational changes; while Asp/Glu and Arg residues may contribute to structural stability, His and Lys residues predominate. Pre-pro-GnRH/GAP is about 30% helix in the range of 2–40°C; however, even at 90°C, the peptide retains nearly 50% of its helix character. There is no evidence for a cooperative transition; for this reason, differential scanning calorimetry failed to yield a defined transition thermogram. Pre-pro-GnRH/GAP apparently does not pass through a transition state as a function of temperature but appears to flex and retain a high percentage of helix structure, resulting in subtle changes in secondary structure. There is no discernible isodichroic point. On either side of the neutralpH range, however, there are dramatic changes in structure that result in nonreversible denaturation of the protein. Relative to N(Ac)Trp-amide, the emission position of intrinsic Trp fluorescence of pre-pro-GnRH/GAP is blue shifted to 338 nm, indicating that the microenvironment(s) encompassing the 2 Trp residues are buried within the protein structure. Synthetic pre-pro-GNRH/GAP is a substrate for GAP-releasing enzyme (the proposed physiologically relevant processing enzyme of the precursor protein) and yields GAP peptide (D14–I69). Of the other serine proteinases tested (trypsin, plasmin, kallikrein), only GAP-releasing enzyme shows this specificity of cleavage. Hierarchical cleavage observed in the time course of proteolysis with trypsin, however, suggests that other peptide products might be formed from GAP once it is processed from the precursor protein by cleavage at sites other than the primary processing site catalyzed by enzymes other than GAP-releasing enzyme. The primary processing site for GAP-releasing enzyme (GLRPGGKR) is thus accessible in the precursor protein, consistent with our hypothesis that the recognition sequence is located at the surface of the protein and acts as a recognition element for the processing endoproteinase. The conformation of the precursor protein is dynamic, supporting the idea that intracellular (and/or intragranular) conditions may play a role in regulation of endoproteolysis. Conformational flexing of the pro-hormone in response to intracellular conditions may serve to differentially expose various processing sites which may help explain tissue specificity of processing.