More than 75% of all proteins are glycoproteins, and it is unlikely that nature undergoes such a biosynthetic effort without a defined need. Yet, the biological function of carbohydrate structures and their biosynthesis is not well understood. A main obstacle had been the lack of suitable analytic techniques to perform structural and functional analyses. With the advent of high sensitivity NMR techniques in the past ten years this scenario has dramatically changed. The analysis of glycosylation patterns of glycoproteins with NMR is therefore an emerging field. We have concentrated on the analysis of human glycosyltransferases since these are the key enzymes that are responsible for specific glycosylation patterns. An understanding of their function will significantly enhance our options to interfere with pathological processes in which glycostructures are involved. At the present we focus on the human blood group B galactosyltransferase (GTB). In collaboration with Prof. Palcic from the Carlsberg Laboratory in Copenhagen we aim at deciphering especially the enzyme dynamics that take place during catalysis. Nothing is known so far about this important issue. Understanding the dynamics of such enzymes at atomic level will greatly enhance the repertoire of approaches to modulate the function of these proteins for biotechnological or pharmaceutical purposes. NMR yields data on the complete time scale of molecular motions from the picosecond into the second time scale. We have also started a collaboration with Prof. Hübner from the Institute of Physics where we correlate NMR data with measurements from single molecule fluorescence spectroscopy. As a first success we have identified the bioactive conformation of donor substrates binding to GTB and we have come to a hypothesis that explains the exquisite specificity with which this enzyme processes its donor substrate. In Fig. 4 we show the dramatic change that donor substrates undergo when binding to GTB, and in Fig. 5 we show how an aspartate and a glutamate act as molecular tweezers to correctly position the donor substrate.

The research projects require access to several techniques:

Protein expression and purification (Dr. Hanne Peters):

Recombinant expression of proteins is routinely performed in E.coli. For NMR experiments with proteins it is required to isotope label the protein with NMR active stable isotopes, i.e. with 13C, 15N and 2H. We have established these techniques in our laboratory and constantly improve the conditions in order to improve the yields of isotope labeled proteins. This is mandatory

since isotope labeling can be extremely costly, especially if 2H and 13C isotopes are involved, or if amino acid selective labeling is performed. We have succeeded in substantially improving known protocols such that e.g.

GTB is produced at concentrations of 100 mg per L of culture. Our current focus is on glycosyltransferases, viral proteases and viral coat proteins.

NMR spectroscopy (Dr. Thorsten Biet)

High resolution NMR spectroscopy allows to analyze (bio)molecules in solution. NMR complements crystallography in that it adds dynamic information to the static picture. Another asset is the ease with which molecular recognition processes can be analyzed by NMR. Therefore, NMR nowadays plays a major role in the analysis of receptor-ligand interactions and in the drug discovery process. We have focused in our projects on carbohydrate-protein recognition processes.

Our NMR instruments include:

• 250 MHz Bruker DRX
• 500 MHz Bruker DRX with TCI cryogenic probe
• 700 MHz Bruker DRX with TXI cryogenic probe
  (located at our outstation at the University of Hamburg)

Biacore and Microcalorimetry (PD Dr. Thomas Weimar)

For a deeper understanding of molecular recognition processes it is important to have access to the thermodynamic signature of such reactions. In our institute surface plasmon resonance experiments (Biacore) are well established and deliver kinetic and thermodynamic information at the same time. As a gold standard we employ microcalorimetry to unravel the thermodynamics of binding events. The combination of Biacore and microcalorimetry has proven to be very powerful. The Institute of Chemistry houses a Biacore 3000 and a Biacore J instrument and has access to an isothermal and differential scanning calorimetry equipment in the Institute of Biochemistry.