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Mechanism of yeast N-glycosylation: Structural characterization of Ost4p and its mutant

Mechanism of yeast N-glycosylation: Structural characterization of Ost4p and its mutant

Name:
Bharat Chaudhary

Department:
Chemistry

Abstract:
N-linked glycosylation of proteins is an essential and highly conserved protein modification reaction that occurs in all eukaryotes. The enzyme that carries out this reaction is called oligosaccharyltransferase (OST). In this reaction, OST modifies the side chain of a specific Asparagine residue with a carbohydrate group during protein synthesis at ribosome. Genetic defects cause a series of disorders that includes but not limited to mental retardation, developmental delay, hypoglycemia etc. Complete loss of N-glycosylation is lethal in all organisms. In yeast, OST consists of nine subunits. OST4p is the smallest subunit. Mutation of Valine (V) at position 23 to Aspartate (D) causes defects in N-glycosylation of proteins. To understand the structure, function and role of Ost4p in this reaction, we are characterizing Ost4p and its mutant by high-resolution solution NMR and circular dichroism (CD) studies. To this end, we have overexpressed, purified 15N, 13C-labeled Ost4p and the mutant OST4p (V23D), and reconstituted in membrane mimetic. The assignments of Ost4p using 3-dimensional NMR, and the preliminary structure calculated will be presented. The 2D {1H, 15N} HSQC spectrum of mutant OST4p (V23D) is significantly different from that of the wild-type suggesting that the mutant has a completely different conformation than the wild-type protein.