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The Skaggs Institute
for Chemical Biology


Scientific Report 2008




Automation of Nuclear Magnetic Resonance Structure Determination of Proteins in Solution

K. Wüthrich, B. Pedrini, P. Serrano, B. Mohanty, R. Horst

We use nuclear magnetic resonance (NMR) spectroscopy in solution for studies in structural biology and structural genomics. The following are
2 illustrations of current applications: structural characterization of the proteome of the coronavirus that causes severe acute respiratory syndrome, which is pursued under the auspices of the Center for Functional and Structural Proteomics of the SARS Coronavirus (FSPS; http://visp.scripps.edu/SARS/default.aspx), and studies of chaperone-mediated protein folding, which is a collaboration with A. Horwich, a guest scientist at Scripps Research from Yale University, New Haven, Connecticut.

In an effort to continually enhance the significance of the NMR observations and the efficiency with which NMR structures can be solved, developing methods is an important part of our activities. During the past year, the team members supported in part or entirely by funds from the Skaggs Institute have made important contributions to new and improved NMR approaches. Because of the important role of NMR in drug discovery and drug design, these developments bear directly on many aspects of biomedical research.

Currently, NMR determinations of protein structure in solution are typically performed by experienced spectroscopists who use interactive informatics tools. Increased use of fully automated steps in structure determination promises to increase the efficiency of the procedure and further add to the reliability of the results obtained. To increase automation of NMR structure determination, a research team directed by me at the ETH Zürich, Zürich, Switzerland, developed new software and new NMR experiments. In the context of our work in structural genomics as part of the Joint Center for Structural Genomics (www.jcsg.org), we have now assembled these software modules into a new protocol for structure determination that includes extensive automation.

In Figure 1 showing the newly introduced automated NMR protocol for structure determination, 2 key features of the procedure are in red. First, a novel approach to quality assessment of the protein solutions intended for NMR structure determination is introduced in the form of the "NMR profile." The NMR profile enables a quantitative assessment of the suitability of the sample for the use of different NMR techniques for assignments of the polypeptide backbone. Second, the protocol includes fully automated structure determination that leads reliably to an accurate determination of the polypeptide backbone fold. Two additional important aspects of the new protocol are as follows: Once the polypeptide backbone assignment has been obtained, the additional information needed for assigning the amino acid side chains and the structure calculation are obtained from the same heteronuclear-resolved [1H,1H]-nuclear Overhauser enhancement spectroscopy (NOESY) data sets, a step that ensures high internal consistency of the entire procedure. The protocol contains 2 important interactive steps to ensure (1) completeness of the polypeptide backbone assignments and (2) refinement and validation of the automatically solved structure.
Fig. 1. Protocol for automated NMR structure determination.

We have so far applied the protocol to a number of different target proteins. As an illustration, Figure 2 shows the NMR structure of the hypothetical protein TM0212 from Thermotoga maritima. For this protein and several other proteins, the automated part of the structure determination, leading to the accurate description of the polypeptide backbone fold (see Fig. 1), was achieved within 1 week.
Fig. 2. Stereoview of the protein TM0212 from T maritima determined by using the protocol of Figure 1. Color scheme: polypeptide backbone, gray; well-structured amino acid side chains, yellow; other amino acid side chains, blue.

Publications

Johnson, M.A., Southworth, M.W., Herrmann, T., Brace, L., Perler, F.B., Wüthrich, K. MR structure of a Klba intein precursor from Methanococcus jannaschii. Protein Sci. 16:1316, 2007.

Pedrini, B., Placzek, W.J., Koculi, E., Alimenti, C., LaTerza, A., Luporini, P., Wüthrich, K. Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the antarctic ciliate Euplotes nobilii. J. Mol. Biol. 372:277, 2007.

Placzek, W.J., Almeida, M.S., Wüthrich, K. NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase. J. Mol. Biol. 367:788, 2007.

Placzek, W.J., Etezady-Esfarjani, T., Herrmann, T., Pedrini, B., Peti, W., Alimenti, C., Luporini, P., Wüthrich, K. Cold-adapted signal proteins: NMR structures of pheromones from the antarctic ciliate Euplotes nobilii. IUBMB Life 59:578, 2007.

 

Kurt Wüthrich, Ph.D.
Cecil H. and Ida M. Green Professor of Structural Biology

Wüthrich Web Site