December 4-7, 2001
Friiberghs Manor, Örsundsbro and Stockholm University
by Kurt Wüthrich
Institut für Molekularbiologie und
Biophysik, Eidgenössische Technische Hochschule Zürich,
CH-8093 Zürich, Switzerland and The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037
USA
The emergence of the new fields of
structural genomics and functional genomics presents us with an
unprecedented availability of new macromolecules with so far
unknown functions and the promise of novel insights into the mode
of action of intact organisms. When focusing on the proteome of
living cells and multi-cellular functional entities, one faces
intricate dynamic phenomena in addition to the abundant wealth of
structural diversity. On the technical level, recent advances in
biochemistry and molecular biology present us with an
ever-increasing array of well-defined functional preparations
from living systems, which are amenable to refined analytical
methods for detailed characterization. The dynamic nature of the
proteome in health and disease, with up- and down-regulation of
individual protein functions and protein concentrations in
response to environmental factors must to a large extent be
related to the dynamic nature of the individual protein
molecules, other classes of compounds with which they interact,
and the rate processes that lead to the formation of transient or
stable functional supramolecular structures. Nuclear magnetic
resonance (NMR) spectroscopy has long had a special position
among modern analytical methods in that it combines atomic
spatial resolution with high temporal resolution in studies of
biological macromolecules.
A first part of this lecture will illustrate the potential of
modern NMR spectroscopy to combine studies of three-dimensional
protein structure with information-gathering on intramolecular
and supramolecular rate processes, and on conformational
transitions in response to diverse extrinsic factors. The systems
studied will include prion proteins from mammalian and
non-mammalian species, the endoplasmatic chaperone system of
calreticulin and ERp57, and the pheromone-binding protein from
Bombyx mori. For these systems the NMR investigations
resulted in de novo protein three-dimensional structure
determinations, as well as in novel insights into the regulation
of protein functions by intramolecular conformational equilibria
or by intermolecular interactions in supramolecular
structures.
One of the demands for analytical methods in the post-genomic era
is that they should be able to cover a wide range of
macromolecular sizes. In this regard the potentialities of NMR
spectroscopy have in recent years been greatly enhanced by the
introduction of transverse relaxation-optimized spectroscopy
(TROSY) and cross-correlated relaxation-enhanced polarization
transfer (CRINEPT). The principles of TROSY and CRINEPT have been
introduced into a wide variety of multi-dimensional NMR
experiments, which now enable sequential resonance assignments in
structures of molecular weights up to about 150 kDa, and the
recording of correlation spectra with structures of size 500 kDa
and beyond. Applications of these techniques will be illustrated
with a structure determination of the E. coli membrane
protein OmpX reconstituted in water-soluble micelles, and with
studies of the E. coli chaperone system of GroEL and
GroES. These results will provide a basis for discussions on the
role of NMR spectroscopy in the anticipated future evolution of
structural and functional genomics, with special emphasis on NMR
as a structural and analytical tool in biomedical research and
drug discovery.
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