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Updated: Jun 12, 2026

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization
Published on: March 16, 2020
Deglycosylated milin unfolds via inactive monomeric intermediates
Subhash Chandra Yadav1, N K Prasanna Kumari, Medicherla V Jagannadham
1Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, UP, India. subhash@ihbt.res.in
Abstract:
The effect of deglycosylation on the physiological and functional organization of milin was studied under different denaturizing conditions. Trifluoromethanesulfonic acid mediated deglycosylation resulted in insoluble milin, which was found to be soluble only in 1.5 M GuHCl with native-like folded structure. Kinetic stability, proteolytic activity, and dimeric association were lost in deglycosylated milin. Urea-induced unfolding revealed two inactive, highly stable equilibrium intermediates at pH 7.0 and pH 2.0. These intermediates were stable between 5.5-6.5 and 5.0-6.0 M total chaotropes (urea + 1.5 M GuHCl) at pH 7.0 and pH 2.0, respectively. GuHCl-induced unfolding was cooperative and noncoincidental with a broad transition range (2.0-5.0 M) at pH 7.0 and pH 2.0. Equilibrium unfolding of deglycosylated milin by urea and GuHCl substantiates the involvement of various inactive monomeric intermediates. This study provides a way to understand the role of glycosylation in the unfolding mechanism, stability, and functional activity of the serine protease milin.
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