Chemistry Colloquium: Dr. Gabriel Cook

The Chemistry Department in the College of Arts and Sciences is pleased to welcome Dr. Gabriel Cook, an Assistant Professor at Oklahoma State University.

Title: In Vitro Glycosylation of Membrane Proteins Involve in Human Disease

Abstract

Glycoproteins take part in nearly every biological process and make up a large percent of the proteome. N-glycosyltransferase (NGT) from Actinobacillus pleuropneumoniae, which recognizes the consensus amino acid sequence, -Asn-X-Ser/Thr- (NXT) within the protein, has been shown to successfully glycosylate peptides and proteins in vitro. The enzyme catalyzes glycosidic bond formation between the oligosaccharide donor, containing nucleoside phosphatase, and the amide nitrogen of the asparagine residue. We are specifically interested in the properties of membrane glycoproteins, which are key components in a number of different disease states. In vitro studies of N-linked glycosylation were done in a step-wise fashion in a membrane mimetic environment to confirm the methods for glycosylating soluble proteins could be applicable to membrane proteins. Detergent and lipid systems were used to solubilize the hydrophobic peptides and membrane proteins for glycosylation. Gel electrophoresis, mass spectrometry, and NMR studies were used for the detection and quantification of glycosyltransferase activity. Our experiments demonstrated that full-length membrane proteins that contain a N-glycosylation consensus sequence, can be glycosylated by NGT, even in the presence of membrane mimetic environments. These methods will be used as the starting point for further oligosaccharide attachment and characterization of structure, dynamic and interactions of these important proteins.