Structural Characterization of Glycosylated Proteins: an NMR Approach

James H. Prestegard

Proteins of mammalian origin include those post-translational modified by the addition of complex carbohydrates (glycoproteins). This class of proteins is not small; it is suggested to include as many of half of all mammalian proteins and nearly all proteins prepared for therapeutic intervention in disease. Abnormal glycosylation has also been directly linked to a number of human diseases including cancer, diabetes, and acute inflammatory response. Yet, in the protein data bank (PDB), less than 1% of the structures deposited actually include coordinates for even the first sugar of an added carbohydrate. There are some reasons for the lack of structural information. The heterogeneity of glycosylation is known to contribute to the failure of glycoproteins to form crystals suitable for study by X-ray diffraction, and the lack of glycosylation in bacterial expression hosts prevents use of this most valuable tool for the production of the isotopically labeled proteins needed for studies by Nuclear Magnetic Resonance (NMR). Methods developed in the NCRR supported Resource for Integrated Glycotechnology are addressing the problems that prevent using NMR as a structural tool. In particular, they combine protein expression in mammalian cell culture with novel single amino acid labeling strategies, new NMR resonance assignment strategies, and new sources of long-range structural constraints to provide a route to glycoprotein structure.

Below some preliminary results on the sialyltransferase ST6Gal 1 are presented. The protein has been labeled with the single amino acid, 15N phenylalanine, a spin-labeled analog of the sialic acid donor, CMP-NeuAc has been synthesized, and the perturbations of NMR resonances on binding this analog have been used to estimate structurally useful distances within the protein. The model illustrated is based on threading a distant homolog. It forms the basis for further steps in structure refinement.

Figure: ST6Gal 1 preliminary results. The position of the enzyme's phenylalanines are shown relative to one of it's substrates.