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The disulphide bond structure of thyroid-stimulating hormone beta-subunit
W D Fairlie1, P G Stanton, M T Hearn
1Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
The Biochemical Journal
|March 1, 1996
Summary
This study maps all six disulfide bonds in bovine thyrotropin beta-subunit (bTSH beta) using a novel double-alkylation strategy and HPLC peptide mapping. This method precisely locates bTSH beta disulfide bonds, advancing protein structure analysis.
Area of Science:
- Biochemistry
- Protein Chemistry
- Structural Biology
Background:
- The beta-subunit of bovine thyrotropin (bTSH beta) contains six disulfide bonds, but only one was previously well-characterized.
- Understanding the precise disulfide bond arrangement is crucial for elucidating protein structure and function.
Purpose of the Study:
- To unequivocally assign the locations of all six disulfide bonds within the bTSH beta subunit.
- To develop and validate a sensitive method for mapping disulfide bonds in proteins with multiple cysteine residues.
Main Methods:
- A double-alkylation strategy using a fluorescent reagent, 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulphonic acid, was employed after selective reduction of bTSH beta.
- Reversed-phase High-Performance Liquid Chromatography (RP-HPLC) peptide mapping and associated structural analyses were used to identify labelled cysteine residues.
- High-performance ion-exchange chromatography was utilized to isolate partly reduced bTSH beta derivatives for further analysis.
Main Results:
- The most reactive disulfide bond was identified as Cys88-Cys95.
- Disulfide bonds Cys16-Cys67 and Cys19-Cys105 were assigned based on the reactivity of involved cysteine residues.
- The previously uncharacterized disulfide bond Cys31-Cys85 was assigned, and the remaining Cys2-Cys52 and Cys27-Cys83 bonds were assigned by homology to human chorionic gonadotropin.
- The study successfully mapped all six disulfide bonds in bTSH beta.
Conclusions:
- The developed double-alkylation strategy combined with RP-HPLC peptide mapping is highly effective for assigning disulfide bonds in proteins like TSH beta, even with limited sample quantities.
- This method provides a robust approach for detailed structural analysis of proteins containing numerous cysteine residues.