POWRE: New Approaches for Efficient Determination of Protein Structures by Solid-State NMR and Isotopic Labeling

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9870373HongMembrane proteins and fibrous proteins constitute more than a third of all proteins and carry out many important biological functions. Yet they cannot be characterized by single-crystal X-ray diffraction and solution-state nuclear magnetic resonance (NMR) due to their noncrystalline and insoluble nature. Solid-state NMR is a powerful probe of the three-dimensional structure of these molecules, but to date it is inefficient for protein structure determination, since normally only a small number of spin labels (e.g. 13C and 15N) can be incorporated into the protein to measure a few molecular structural parameters at a time. The goal of this research is to develop a novel isotopic labeling approach and combine it with multi-dimensional solid-state NMR to obtain many structural parameters per experiment, thus elucidating protein structure more efficiently. This labeling scheme will exploit the biosynthetic pathways of amino acids to achieve selective but extensive enrichment of 13C. This will create many spin pairs for internuclear distance measurements while reducing the complex multi-spin interactions present in uniformly 13C-labeled proteins. This labeling approach will be tested on a well-characterized and stable protein, ubiquitin. Based on this labeling scheme, two types of solid-state NMR experiments will be explored. The first type of experiment probes the protein secondary structure by measuring the backbone torsion angles f and y. The second type of experiment constrains the protein tertiary structure from internuclear distances, especially long-range distances between the 15N and 13C on the side chains of different residues.While trained in the area of NMR throughout her professional life, Dr. Hong's interest has been understanding the structure of small peptides. With POWRE support, she intends to move into the arena of large polypeptides, employing molecular biological techniques of protein biosynthesis and isotopic labeling. The support will provide her the needed start-up for the proposed study, and allow her to expand her teaching into areas of advanced NMR and biophysics of macromolecules to both undergraduate and gradute students at U Mass. With the support, Dr. Hong also intends to organize a number of workshops on introductory NMR and two-dimensional NMR for biomolecular structure determination.