Effects of Glycosylation on Protein Structure, Function and Dynamics
Even though more than half of all human proteins are glycosylated, the functional characteristics of this important posttranslational modification is so far poorly understood and structural information is rare. One obstacle to investigate these proteins is due to the fact that they can most often not easily be overexpressed in Escherichia coli. Obtaining the correctly modified protein requires more complicated large-scale recombinant eukaryotic expression systems (such as insect cells). Furthermore, the size, flexibility and heterogeneity of these proteins are a challenge for the analysis and structure determination.
By using NMR spectroscopy, we aim to provide information about the changes in the protein structure and dynamics (to be elucidated at atomic resolution) upon glycosylation, also when the target protein is unstructured or when multiple conformations are present. This requires the development of specific isotope labeling protocols for the recombinant expression of glycosylated proteins in eukaryotic hosts. The initial focus is on the C-mannosylation of tryptophan residues in TSR1, the thrombospondin type 1 repeat from the netrin receptor UNC-5. The addition of a hydrophilic mannose undoubtedly affects the localization and characteristics of the tryptophan thereby modulating the protein properties. This modification possibly plays a key role in the function of the protein. Ligand binding, receptor internalization, proper folding, export, signal transduction and cell migration have been suggested to be affected by C-mannosylation. Our studies can be expanded to multi-domain interactions of mannosylated proteins and the research focus can be broadened by adding new systems, such as extracellular domains of growth factor receptors.
Team
Harald Schwalbe (Pi) is interested in understanding the influence of glycosylation on the structure, function and dynamics of proteins mainly by solution state NMR but also by other structural techniques (such as X-ray crystallography and Small-Angle X-ray Scattering (SAXS)), The group has expertise on structural investigation of soluble glycosylated extracellular domains (ECD) of receptors with their hormones, which was gained during a research collaboration with the pharmaceutical company Sanofi.
Krishna Saxena is committed to the development of of specific isotope labeling protocols for the expression of glycosylated and non-glycosylated proteins.
Henry Jonker has expertise in experimental NMR investigations, analysis, assignment and has elucidated the structure and studied the dynamics of various biomolecules (proteins, RNA, DNA, Ligands) and their complexes by NMR.
For more information on Harald Schwalbe’s lab go to his website.
Collaborations
P1 Hans Bakker – Thrombospondin type 1 repeat (TSR1)-containing proteins (UNC-5, MIG-21)
P7 Irmgard Sinning – NMR studies of the yeast Pmt2 construct
