Browsing by Author "Subhash Chandra Yadav"

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A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties
(2009) Subhash Chandra Yadav; M.V. Jagannadham; Suman Kundu; Medicherla V. Jagannadham
Milin, a potent molluscicide from the latex of Euphorbia milii, holds promise in medicinal biochemistry. Electrophoresis, size exclusion chromatography, mass spectrometry and other biochemical characteristics identify milin as a homodimeric, plant subtilisin-like serine protease, the first of its kind. The subunits of milin are differentially glycosylated affecting dimer association, solubility and proteolytic activity. The dimeric dissociation is SDS-insensitive and strongly temperature dependent but does not appear to be linked by disulfide bridges. N-terminal sequence of acid hydrolyzed peptide fragments shows no homology to known serine protease. Peptide mass fingerprinting and de novo sequencing of the tryptic fragments also did not identify putative domains in the protein. Milin seems to be a novel plant enzyme with subunit association partly similar to human herpes virus serine proteases and partly to penicillin binding proteins. Its behaviour on SDS-PAGE gels and other properties is like "kinetically stable" proteins. Such subunit association and properties might play a critical role in its physiological function and in controlling Schistosomiasis. © 2008 Elsevier B.V. All rights reserved.
Complete conformational stability of kinetically stable dimeric serine protease milin against pH, temperature, urea, and proteolysis
(2009) Subhash Chandra Yadav; Medicherla V. Jagannadham
Spectroscopic, calorimetric, and proteolytic methods were utilized to evaluate the stability of the kinetically stable, differentially glycosylated, dimeric serine protease milin as a function of pH (1.0-11.0), temperature, urea, and GuHCl denaturation in presence of 8 M urea at pH 2.0. The stability of milin remains equivalent to that of native at pH 1.0-11.0. However, negligible and reversible alteration in structure upon temperature transition has been observed at pH 2.0 and with 1.6 M GuHCl. Irreversible and incomplete calorimetric transition with apparent Tm > 100°C was observed at basic pH (9.0 and 10.0). Urea-induced unfolding at pH 4.0, and at pH 2.0 with GuHCl, in presence of 8 M urea also reveals incomplete unfolding. Milin has been found to exhibit proteolytic resistant in either native or denatured state against various commercial proteases. These results imply that the high conformational stability of milin against various denaturating conditions enable its potential use in protease-based industries. © 2009 European Biophysical Societies' Association.
Deglycosylated milin unfolds via inactive monomeric intermediates
(2010) Subhash Chandra Yadav; N.K. Prasanna Kumari; Medicherla 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. © 2010 European Biophysical Societies Association.
Equilibrium unfolding of kinetically stable serine protease milin: The presence of various active and inactive dimeric intermediates
(Springer Verlag, 2010) Subhash Chandra Yadav; Medicherla V. Jagannadham; Suman Kundu
Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has a-helix and β-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.
Uwebraunomyces, a novel genus of the family Mycosphaerellaceae
(Westerdijk Fungal Biodiversity Institute, 2025) Gurdip Singh; Priyanka Kumari; Soumyadeep Rajwar; Subhash Chandra Yadav; Smriti Mall; S. Saran Kumar; Ravindra Nath Singh
A hyaline to pale olivaceous brown hyphomycetous fungus was collected on living leaves of Tinospora cordifolia in India. Molecular phylogenetic analyses of several loci (LSU-RPB2-ITS) indicated a relationship with cercosporoid fungi (Mycosphaerellaceae). As no other known lineage shares similar morphology or DNA sequences, a new genus, Uwebraunomyces, is proposed, with U. tinosporae comb. nov. based on Ramularia tinosporae as the type species. Conidiophores are pale olivaceous to very pale brown in colour, typically unbranched, and subcylindrical to geniculate-sinuous at the tip. Conidia are solitary, obclavate to obpyriform, straight to curved, and few-septate. Additionally, the conidiogenous loci are characterized by a centrally located small papilla and a minute, distinct frill that is typically evident in older scars. These characteristics distinguish Uwebraunomyces from other closely related members of the ramularioid complex. Additionally, Cercosporella bundelkhandae is recognized as a new synonym as it shares nearly identical morphological features with U. tinosporae. At the ultrastructural level, Uwebraunomyces is further characterized by conidiogenous loci that bulge outward, forming convex or truncated cone shapes, with wall thickening extending beyond the area originally occupied by the base of the conidium. Older scars display a minute papilla or small, somewhat conical protuberant like structure in the centre of their apical depression, often accompanied by a minute, distinct frill. The conidial base is obconically truncate, featuring a basal depression with a centrally located, slightly protuberant structure. These distinct ultrastructural features set Uwebraunomyces apart from other members of the ramularioid complex, underscoring its taxonomic significance within the Mycosphaerellaceae. © 2025 Westerdijk Fungal Biodiversity Institute.