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Deglycosylated milin unfolds via inactive monomeric intermediates

journal contribution
posted on 2010-11-01, 00:00 authored by Subhash Chandra Yadav, N K Prasanna Kumari, M V Jagannadham
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.

History

Journal

European biophysics journal

Volume

39

Issue

12

Pagination

1581 - 1588

Publisher

Springer

Location

Heidelberg, Germany

ISSN

0175-7571

eISSN

1432-1017

Language

eng

Publication classification

C1.1 Refereed article in a scholarly journal

Copyright notice

2010, European Biophysical Societies’ Association