By Jason Socrates
is no greater pleasure for me than to practice and exhibit
van Beethoven, 1800.
Even with the most powerful and sensitive of modern nuclear
magnetic resonance (NMR) instruments, many interesting systems
are impossible to solve because they are simply too big.
And structural biologists have sought for ways in which
to breach, stretch, surpass, or otherwise blow away this size
limitation. Some biologists have wanted to do this for decades.
"We have submitted proposals as early as 1979 to do basic
research on ways to overcome the physics barrier to doing
solution NMR on large structures," says Kurt Wüthrich,
who is currently guest scientist in the Department of Molecular
Biology at The Scripps Research Institute (TSRI) and is scheduled
to become a full-time faculty member in 2004. "Methods development
is an important direction of my laboratory."
In fact, since the 1960s, Wüthrich has pioneered the
field of NMR structure determination. He is professor of biophysics
and former chair of the department of biology at the Eidgenössische
Technische Hochschule (ETH) in Zürich, Switzerland, where
he was involved in the first de novo protein structure
determinations with NMR. And he is author of one of the definitive
books on the subject, "NMR of Proteins and Nucleic Acids"
But until recently, even Wüthrich had to accept size
limits for studies of biological macromolecules in solution
The Trouble With Large Systems
"It used to be that there was a molecular weight limit well
below 50,000 daltons for the use of solution NMR," says Wüthrich
(A dalton is equivalent to the mass of one hydrogen atom),
and this was not an engineering limit, but a physics one.
Large structures such as many protein/protein, protein/DNA
or protein/lipid complexes are impossible to investigate with
conventional NMR because of their slow response to the thermal
motion of the solvent molecules in the test tube.
Proteins in solution are constantly bombarded with solvent
molecules and undergo random "Brownian" motion as a result.
The larger the structure is, the more slowly it will respond
to the impact and the more slowly it will reorient itself
in solution. This slow response deteriorates NMR spectra and
makes structure determination impossible.
"This we have overcome by using single-transition spectroscopy,"
says Wüthrich, referring to the new technique of transverse
relaxation-optimized spectroscopy (TROSY).
TROSY is a technique that suppresses the transverse nuclear
spin relaxationthe event that causes the deterioration
of NMR spectra for larger molecular structures.
For any two coupled "NMR atoms" (see: Primer on NMR of Biomolecules)
in a molecule, there will be a total of four energy transitions
that can be detected by NMR. Conventional NMR averages all
four, but this averaging deteriorates the signal when the
molecules get too large. TROSY selects only one of the four
transitionsthe one that is insensitive to Brownian motion
in a high magnetic field.
The optimal frequency for the TROSY effect is calculated
to be near 1000 MHz, and since TSRI's new 900 MHz NMR is closer
to this than any instrument ever before, Wüthrich will
be able to effectively apply TROSY to very high molecular
weight systems on the new instrument.
With TROSY, the size limit of structures that can be solved
with NMR is expanded several-fold. "We can now do reasonably
detailed structural investigations of proteins in structures
of size up to about 150,000 daltons," says Wüthrich,
who was the first to recognize and exploit TROSY at ultra-high
magnetic field strengths with his research group in Switzerland.
In structures that are even larger than 150,000 daltons,
though, TROSY is no longer effective on its own. But Wüthrich
has pioneered other techniques, such as cross-correlated relaxation-enhanced
polarization transfer (CRINEPT). When combined with TROSY,
these result in highly effective experiments for very large
"We can go up to one million [daltons], essentially," he
Solving Structures Great and Small
Besides developing methodology, much of Wüthrich's
research is devoted to solving novel structures of biological
molecules, and he works on many systems, from the very small
to the very large.
He studies pheromone molecules and their receptors from
insects and other organisms, for instance. Pheromones may
be small organic molecules or very stable proteins of about
35 to 50 residues, which transmit a behavioral stimulus, often
sexual. Animals emit pheromones in minute amounts and have
receptor systems that can detect these.
Wüthrich has solved numerous protein pheromone structures
from Mediterranean sea creatures and pheromone-binding proteins
from other organisms, such as silkworms.
He also has a program looking at ways of solving membrane
protein structures. Of all the relevant molecular structures
in biology, membrane proteins have been solved the leastmainly
because they are so hard to work withand there is a
great many membrane proteins that need to be solved. Less
than one half of one percent of the structures contained in
the Brookhaven National Laboratory Protein Data Bank are of
integral membrane proteins, despite the fact that over a third
of all proteins in the body are in the membrane.
Wüthrich's group studies the recombinant expression,
reconstitution, and isotope labeling of integral membrane
proteins for use in NMR. These are not particularly large
proteins, typically 200 residues, but the experiments require
the proteins to be suspended in large soap-like micelles,
which mimic the membrane and preserve the fold of the proteins.
"You can't do conventional NMR on such things," says Wüthrich,
"only TROSY enables such projects."
Wüthrich also is looking at chaperonin systems like
GroELthe very large protein complex that monitors the
folding of newly synthesized proteins.
GroEL is a multimeric protein whose subunits14 of
themcome together to form two 7-subunit toroids that
stack together. This 800,000 dalton structure binds misfolded
proteins, which fit into the inside cavity of the toroid shape
where they can refold. Since GroEL is such a large system,
it cannot be studied by conventional NMR.
Combining CRINEPT and TROSY, however, says Wüthrich,
"We can do GroEL and we have observed other, smaller proteins
when bound to GroEL."
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