NMR spectroscopic investigation of a chaperone complex

The three-dimensional structure of a chaperone complex in aqueous solution will be studied using nuclear magnetic resonance (NMR) spectroscopic methods. Chaperones are proteins that assist unfolded or incorrectly folded proteins to attain their folded, native state. Chaperones often form part of a network of specific folding factors that acts as a quality control system to ensure that the desired, properly folded and biologically active form of proteins is produced. The goal of this project is the structural investigation of a complex comprised of two chaperone molecules that is located in the endoplasmic reticulum. This chaperone complex is involved in the assisted folding of glycoproteins, including various cell membrane receptors, trans membrane channels and serum proteins. It consists of the glycoprotein binding chaperone calreticulin and a multi-domain chaperone and protein disulfide isomerase, ERp57. The size of the protein complex that will be studied within this project reaches the upper limit of biological macromolecules amenable to NMR spectroscopic studies using methods that are currently available. In collaboration with the laboratories of Prof. Robert Konrat at the University of Vienna and Prof. Lewis E. Kay at the University of Toronto the existing NMR spectroscopic methodology will be further optimized for studying proteins of high molecular weight. For the assignment of resonances, a series of triple-resonance three- and four- dimensional NMR experiments will be performed on protein samples enriched in NMR active isotopes (15N, 13C, 2H). Structural information will be obtained from a variety of anisotropic and isotropic interactions, such as dipolar couplings, chemical shift anisotropy, nuclear Overhauser effects and isotropic chemical shifts. The long-range nature inherent to dipolar couplings is particularly useful for defining relative orientations of individual components in multi-domain proteins and protein complexes such as ERp57/calreticulin. Using this approach, the three- dimensional assembly of the chaperone complex, i.e. the orientation of the individual protein domains in ERp57 as well as the orientation of the two chaperone molecules in the complex with respect to each other, will be determined. In addition, dynamic properties of the chaperones will be investigated employing relaxation measurements. Since the biological function of proteins is closely related to their three-dimensional structure and dynamic properties, the results from this study will provide new insights into the mechanism of assisted protein folding.