Structure and mechanism of an active lipid-linked oligosaccharide flippase.

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK.

On the efficiency of NHS ester cross-linkers for stabilizing integral membrane protein complexes.

We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes.

A catalytically essential motif in external loop 5 of the bacterial oligosaccharyltransferase PglB.

Asparagine-linked glycosylation is a post-translational protein modification that is conserved in all domains of life. The initial transfer of a lipid-linked oligosaccharide (LLO) onto acceptor asparagines is catalyzed by the integral membrane protein oligosaccharyltransferase (OST). The previously reported structure of a single-subunit OST enzyme, the Campylobacter lari protein PglB, revealed a partially disordered external loop (EL5), whose role in catalysis was unclear.

Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation.

The initial glycan transfer in asparagine-linked protein glycosylation is catalysed by the integral membrane enzyme oligosaccharyltransferase (OST). Here we study the mechanism of the bacterial PglB protein, a single-subunit OST, using chemically synthesized acceptor peptide analogues. We find that PglB can glycosylate not only asparagine but also glutamine, homoserine and the hydroxamate Asp(NHOH), although at much lower rates.

Mechanism of bacterial oligosaccharyltransferase: in vitro quantification of sequon binding and catalysis.

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined.