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Human gene 2 relaxin chain combination and folding
Jian-Guo Tang1, Zhao-Hui Wang, Geoffrey W Tregear
1National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Biochemistry
|March 5, 2003
Summary
Relaxin folding involves a stepwise process where the A-chain
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- Relaxin, a peptide hormone from the insulin superfamily, is crucial for connective tissue remodeling during childbirth.
- Its production involves oxidative folding and proteolytic processing, similar to insulin, but its folding pathway is less understood.
- Understanding relaxin's folding is key to elucidating its biological function and potential therapeutic applications.
Purpose of the Study:
- To investigate the folding pathway of relaxin, focusing on the formation of its intramolecular disulfide bonds.
- To compare the oxidation behavior of native relaxin A-chain with synthetic cysteine analogues.
- To determine the role of the A-chain's intramolecular disulfide bond in the overall folding and combination with the B-chain.
Main Methods:
- Synthesis of pairwise serine-substituted relaxin A-chain cysteine analogues.
- Study of oxidation behavior of A-chain analogues alone and with native B-chain.
- Circular dichroism spectroscopy to analyze secondary structure.
- Analysis of A-chain and B-chain combination efficiency under varying conditions.
Main Results:
- Native relaxin A-chain rapidly oxidized to a bicyclic product, while individual disulfide bond formation was slower.
- A non-native isomeric disulfide bond (Cys11-Cys15) formed rapidly, suggesting it may be a folding intermediate.
- The intramolecular disulfide bond in the A-chain is essential for efficient combination with the B-chain.
- Relaxin formation occurs in a stepwise manner, analogous to insulin folding and chain combination.
- Combination efficiency was not significantly affected by temperature, and preoxidized A-chain combined well with S-reduced B-chain.
Conclusions:
- Relaxin folding is a stepwise process initiated by the formation of the A-chain's intramolecular disulfide bond.
- The rapid formation of the Cys11-Cys15 disulfide suggests a potential intermediate in the native folding pathway.
- Relaxin can be efficiently synthesized by combining preoxidized A-chain with S-reduced B-chain, offering a viable method for production.