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Conformational analysis and proteolytic processing of synthetic pre-pro-GnRH/GAP protein
J L You1, S C Milton, R C Milton
1Virginia Commonwealth University, Department of Biochemistry and Molecular Biophysics, Richmond 23298-0614.
Journal of Protein Chemistry
|April 1, 1993
Summary
Synthetic pre-pro-GnRH/GAP protein inhibits prolactin release, a novel finding for a precursor protein. Its conformational properties reveal thermal stability but pH sensitivity, with a high helical content across temperatures.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Pre-pro-GnRH/GAP protein, synthesized via solid-phase methods, is typically retained within the endoplasmic reticulum.
- Precursor proteins generally lack significant biological activity.
- This study investigates the conformational properties of synthetic pre-pro-GnRH/GAP.
Purpose of the Study:
- To characterize the conformational properties of synthetic pre-pro-GnRH/GAP protein.
- To establish a foundation for determining its solution phase conformation using NMR.
- To correlate conformational stability with its observed biological activity.
Main Methods:
- Synthesis of homogeneous pre-pro-GnRH/GAP protein (100 mg) using solid-phase methods.
- Spectroscopic analysis including thermal and pH titration fluorescence and circular dichroism.
- Differential scanning calorimetry (DSC) to assess thermal transitions.
Main Results:
- Synthetic pre-pro-GnRH/GAP exhibits significant biological activity by inhibiting prolactin release from pituitary cells.
- The protein is resistant to thermal denaturation, retaining ~50% helical content even at 90°C.
- Pre-pro-GnRH/GAP is highly sensitive to pH changes, undergoing nonreversible denaturation outside the neutral pH range.
Conclusions:
- This is the first demonstration of significant biological activity in a precursor protein (pre-pro-GnRH/GAP).
- The protein's conformational stability is primarily influenced by pH rather than temperature.
- Amino acid residues like His and Lys play a predominant role in structural stability, alongside Asp/Glu and Arg.