Asparagine-linked glycosylation, catalyzed by the enzyme oligosaccharyl transferase (OT), is an essential protein modification reaction associated with all eukaryotic and some prokaryotic organisms.
The specific aims of this proposal include: 1. Heterologous Expression, Protein Biochemistry and Reconstitution of Bacterial and Eukaryotic OT: Studies are aimed at understanding the unique specificity for glycosylation in the Asn-Xaa-Ser/Thr tripeptide recognition sequence in different organisms through a detailed comparison of the bacterial and eukaryotic enzymes. Research will focus on establishing the activity of the bacterial enzyme in a defined biochemical assay. These studies will set the stage for understanding the mechanism of glycosylation in a simple monomeric system and for elucidating the role of SttSp in OT function in a representative eukaryote (S. cerevisiae). The detailed analysis of the bacterial enzyme will provide a simple working platform upon which to build the research efforts with the more complex eukaryotic enzyme. 2. Development of Selective Inhibitors of Bacterial OT:
This specific aim focuses on the synthesis and in vitro and in vivo evaluation of selective, bioavailable inhibitors of prokaryotic OT. Examination of prokaryotic and eukaryotic glycosylation sequences suggests that there may be considerable differences between the extended-binding site specificities of enzymes from different species, therefore the potential for developing species-selective inhibitors is excellent. Development of more stable, non-peptidyl inhibitors, through the implementation of non-peptidic scaffolds or peptidomimetic isosteres will follow after the identification of a prototype inhibitor. The species selectivity of the inhibitors will also be evaluated using the newly developed eukaryotic cellular assay for OT. 3. Investigation of the effect of N-linked glycosylation on the kinetics of folding of small proteins: In this specific aim we shall develop synthetic and semi-synthetic methodologies for the preparation of a small, homogeneously glycosylated proteins for evaluating the specific effects of glycosylation on the pathway and kinetics of protein folding through detailed biophysical analysis.
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