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July 15, 2002
Volume 80, Number 28
CENEAR 80 28 p. 9
ISSN 0009-2347


SCIENCE

HUGE PROTEIN ANALYZED BY NMR
Ability to study large NMR-accessible biomolecules skyrockets

STU BORMAN

Increasingly sophisticated nuclear magnetic resonance spectroscopy (NMR) techniques have made it possible to analyze larger and larger biomolecules in the past few years. But structures exceeding 100 kilodaltons have remained extremely difficult or impossible to study by NMR.

Now researchers have made nearly an order of magnitude leap in the mass range of proteins amenable to NMR analysis by analyzing the GroEL-GroES chaperone system, a huge 900-kDa protein complex. Chaperones help proteins fold into their native states.

CHAPERONE In the GroEL-GroES complex (shown here in surface and cross-sectional representations), GroES (white) and both halves of GroEL (multicolored and gold) are each made up of seven identical subunits (highlighted in multicolored segment of GroEL).
BASED ON A CONCEPT BY THE LATE PAUL B. SIGLER
The analysis was carried out by molecular biophysics professor Kurt Wüthrich and postdoc Jocelyne Fiaux at the Swiss Federal Institute of Technology (ETH), Zurich, in collaboration with genetics professor and Howard Hughes Medical Institute investigator Arthur L. Horwich and postdoc Eric B. Bertelsen at Yale University School of Medicine [Nature, 418, 207 (2002)]. They accomplished the feat by combining the principles of two high-mass NMR techniques developed earlier by Wüthrich's group: transverse relaxation-optimized spectroscopy (TROSY) and cross-correlated relaxation-enhanced polarization transfer.

"The sound barrier has been shattered big-time in terms of what can be looked at with NMR," Horwich says. "This initial study is more proof of principle, but it suggests we will be able to analyze the [GroEL-GroES] system in a way that's never been possible before."

Wüthrich, Horwich, and coworkers did not determine the NMR solution structure of GroEL-GroES. Instead, they observed resonances that serve as markers for a significant number of individual protons in the complex, which is much less difficult. But these markers do make it possible to study the protein's local dynamic and structural properties--such as the responses of specific amino acid residues when GroES binds to GroEL. (The major structural breakthrough on GroEL-GroES was actually achieved in 1997, when Horwich's group, along with that of the late Yale biochemistry professor Paul B. Sigler, nailed down the complex's structure using X-ray crystallography.)

The GroES-GroEL system is a favorable case for extremely high mass NMR analysis. GroES has seven identical subunits and GroEL has 14, so the number of unique NMR resonances in GroEL-GroES is much lower--and the effective concentration of each subunit is much higher--than would be the case for a monomeric complex of similar mass.

Nevertheless, "the fact that they can study a system of that size is really fantastic," says NIH biophysical chemist Ad Bax. "It potentially brings a whole lot of problems within NMR's reach that we would not have dared to dream about prior to this work. With ongoing improvements in NMR hardware and sensitivity, it is quite conceivable that similar data could eventually be obtained for monomeric systems."

The development of TROSY "enabled studies of systems up to the 100-kDa range," comments chemistry professor David E. Wemmer of the University of California, Berkeley, but most people in the field did not believe until now "that studies of still bigger complexes were really possible."

Chemistry and biochemistry professor Lewis E. Kay of the University of Toronto says that the ability "to get some information about such a huge system is impressive. But we don't have the techniques to be able to assign all the nuclei in a biomolecule that's this large. This study beautifully illustrates the potential for dissecting out very large systems based on having a priori information on the smaller components that go into them."

8028HorwichBertelsen_1 8028WuthrichFiaux_3
YALE UNIVERSITY PHOTO PHOTO BY RUDOLF BAUMANN, ETH ZURICH
CHAPERONE TEAM Fiaux (left in photo at top right) and Wüthrich, and Bertelsen (left in top left photo) and Horwich.



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Copyright © 2002 American Chemical Society



 
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