Glycoproteins Play an Important Role in Many Recognition, Signaling, Trafficking and Adhesion Processes in All Living Organisms

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Glycoproteins play an important role in many recognition, signaling, trafficking and adhesion processes in all living organisms. Thus, engineering of tailor-made glycoproteins will decisively change our capabilities in influencing and controlling complex biological systems. The detailed understanding of the protein glycosylation process is a prerequisite for this endeavor. S-layer glycoproteins represent a natural self-assembly system enabling high-density surface display of glycans in a nanometer-scaled, periodic way. In the biosynthesis of the S-layer glycoprotein glycan of our model organism Geobacillus stearothermophilus NRS 2004/3a several glycosyltransferases and an ABC-transporter are involved in assembly and export of the glycan chain. But the key module of glycan biosynthesis, and thus, for the envisaged carbohydrate engineering is the oligosaccharyl: protein transferase WsaB, a transmembrane protein, which is responsible for the transfer of the glycan chain to the S-layer protein. In this research project (i) the crystal structure of the oligosaccharyltransferase WsaB of G. stearothermophilus NRS 2004/3a and truncated forms thereof will be determined by X-ray crystallography, (ii) structural insights into the reaction mechanism of WsaB concerning substrate specificity and the influence of conserved amino acids in the catalytic domain should be gained by co-crystallization experiments and site-directed mutagenesis and (iii) the interaction of WsaB with the sequence adjacent to the O-glycosylation sites of the S-layer protein SgsE of G. stearothermophilus NRS 2004/3a will be investigated. Whereas numerous structures of soluble proteins have already been published, only fewer than 200 membrane protein structures are known. Membrane protein crystallography remains a significant technical challenge almost throughout the process from clone to structure. The biggest challenge is the expression and purification of these proteins. The experiments will be performed in the laboratory of Prof. James Naismith at the University of St Andrews (UK), who is specialized in X-ray crystallography of membrane proteins. Thus, a profound knowledge about cloning, expression, purification and crystallization of transmembrane proteins is available in this laboratory. Understanding the functionality of the catalytic domains will allow engineering of the domains that contribute to substrate specificity, thus making it possible to alter the specificity of the oligosaccharyltransferase, which then shall be exploited as a key module for the design of "intelligent" glycoproteins..

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