The Skaggs Institute
for Chemical Biology
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,
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
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.
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.
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.
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.