title: Deglycosylated milin unfolds via inactive monomeric intermediates.
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.
authors.affiliation: Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, UP, India. subhash@ihbt.res.in
authors.forename: Subhash Chandra
authors.initials: SC
authors.lastname: Yadav
authors.forename: N K
authors.initials: NK
authors.lastname: Prasanna Kumari
authors.forename: Medicherla V
authors.initials: MV
authors.lastname: Jagannadham
mesh_terms: D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008698:Mesylates / Q000494:pharmacology; D017510:Protein Folding / Q000187:drug effects*; D017433:Protein Structure, Secondary / Q000187:drug effects; D057057:Serine Proteases / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
chemical_list: D006027:Glycosides; D008698:Mesylates; D014508:Urea; D057057:Serine Proteases; C012077:trifluoromethanesulfonic acid; D019791:Guanidine