How multi-subunit proteins come together to form an active complex is a fundamental question which lies at the center of cellular function. Particularly, the Oligosaccharyltransferase (OT) is a complex enzyme, comprised of at least nine integral membrane polypeptides, which attaches a preassembled oligosaccharide to nascent polypeptides during protein translocation into the endoplasmic reticulum (ER). Although all nine polypeptides are necessary for optimal function in vivo, only four polypeptides are necessary for enzymatic activity in vitro. This proposal is aimed at taking a biophysical approach to understanding and characterizing the intricate network of interactions which bring the four essential subunits of OT together to form an active complex. This will be addressed by preparing each of the domains individually, combining them in a one by one fashion, and characterizing the resulting interactions using fluorescence, circular dichroism, mass spectrometry, analytical ultracentrifugation, surface plasmon resonance, native and SDS PAGE, and gel filtration chromatography. Characterization will be done separately for both the luminal and transmembrane domains in order to disentangle the respective roles. After gaining a map of the organization, focus will turn to the reconstitution of OT using the full length polypeptides in model membranes.
| Chen, Mark M; Glover, Kerney Jebrell; Imperiali, Barbara (2007) From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni. Biochemistry 46:5579-85 |
| Glover, Kerney Jebrell; Weerapana, Eranthie; Chen, Mark M et al. (2006) Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway. Biochemistry 45:5343-50 |
| Glover, Kerney Jebrell; Weerapana, Eranthie; Imperiali, Barbara (2005) In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation. Proc Natl Acad Sci U S A 102:14255-9 |
| Weerapana, Eranthie; Glover, Kerney Jebrell; Chen, Mark M et al. (2005) Investigating bacterial N-linked glycosylation: synthesis and glycosyl acceptor activity of the undecaprenyl pyrophosphate-linked bacillosamine. J Am Chem Soc 127:13766-7 |
