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 reticulum, 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 homonuclear 1H-NMR protocols. Thermal and pH titration fluorescence and circular dichroism spectroscopies reveal that the protein is resistant to thermal-induced conformational changes but is particularly sensitive to pH-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 degrees C; however, even at 90 degrees 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 neutral pH range, however, there are dramatic changes in structure that result in nonreversible denaturation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
You
J L
JL
Virginia Commonwealth University, Department of Biochemistry and Molecular Biophysics, Richmond 23298-0614.
Milton
S C
SC
Milton
R C
RC
Rangaraju
N S
NS
Harris
R B
RB
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism; D005454:Fluorescence Polarization; D020690:GTPase-Activating Proteins; D007987:Gonadotropin-Releasing Hormone / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011498:Protein Precursors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D011498:Protein Precursors; D011506:Proteins; C050866:progonadoliberin I; D007987:Gonadotropin-Releasing Hormone; D010450:Endopeptidases; C052441:gonadotropin-associated-peptide releasing enzyme