CHEMICAL & ENGINEERING
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STRUCTURE
ANALYSIS
NMR STRUCTURES 0F LARGER PROTEINS Isotope labeling of amino acids leads to simpler, less congested NMR spectra
P
R O T E I N PROPERTIES AND
interactions are often studied through nuclear magnetic resonance (NMR) spectrometry, which can provide 3-D solution structures of proteins. But it's currently difficult to elucidate N M R structures oflarge proteins because their spectra are too complex. At
stereo- and regiospecific manner, and a protein is then synthesized from the labeled amino acids. The N M R spectrum of the resulting SAIL protein is simpler, less congested, and more easily interprétable than that of the corresponding conventional protein. Kainosho and coworkers believe t h e ap-
SAILORS Researchers who helped develop the SAIL approach include (from left) Akira Mei Ono, Teppei Ikeya, Tsutomu Terauchi, Kainosho, Mitsuhiro Takeda, Peter Giintert, and Seiji Tsuchiya. present, fewer than 2% ofall N M R structures in the Protein Data Bank have masses exceeding 25 kDa, whereas many proteins weigh in at hundreds of kilodaltons. Stereo-array isotope labeling (SAIL) could now ameliorate this problem. Chemistry professor Masatsune Kainosho of Tokyo Metropolitan University (TMU) and coworkers, with support from the Japan Science & Technology Agency, developed the technique and report its use to determine the structure of a 41-kDa protein (Nature 2006,440,52). In SAIL, chiral organic synthesis is used to prepare amino acids labeled with deuterium, carbon13, and nitrogen-15 in a highly WWW.CEN-0NLINE.ORG
proach should make it possible to routinely solve the structures of proteins at least twice as large as those commonly determined today by NMR. Drawbacks are that the labeled amino acids are difficult to synthesize and must be incorporated into proteins by in vitro cell-free protein synthesis, which is not easy to carry out. But a TMU-based venture company, with Japanese government support, is being set up to supply SAIL amino acids commercially. Kainosho and coworkers are also trying to fully automate SAILbased structure determination and to push the technique's molecularweight limits ever higher. SAIL is "a triumph and t h e
achievement of a lifetime," comments Kurt W ù t h r i c h , a professor of molecular biology and biophysics at the Swiss Federal Institute of Technology, Zurich, who shared the 2002 Nobel Prize in Chemistry for work on N M R structure analysis. "It's difficult to estimate just how far it could go, but it could really ring in a new era" in protein NMR. In addition to permitting larger proteins to be analyzed, SAIL could also enable some N M R experiments that Kainosho aren't practical currently, assuming "SAIL amino acids become widely available at an affordable price," Wùthrich notes. With the same proviso, SAIL "is going to have major impact on structural protein N M R studies," says Ad Bax, section chief of the National Institute of Diabetes & Digestive & Kidney Diseases, Bethesda, Md. It's "an extremely important and long-sought advance," according to professor of molecular biology Peter E. Wright of Scripps Research Institute, Lajolla, Calif. "It's a real tour de force and a major breakthrough. The degree of spectral simplification and the reduction in line width and resonance overlap that can be achieved by SAIL labeling are very impressive and promise to revolutionize N M R structure determination for large proteins of 40 kDa and above." Biochemistry professor John L. Markley of the University of Wisconsin, Madison, a collaborator of Kai nosho's, adds that SAIL "represents the culmination of over 10 years of hard work" by Kainosho's group and "has NOT TOO BIG FOR NMR much forward potential" for Kainosho and coworkers large-protein analysis, solid- used the isotope-labeling state N M R , and "answer- technique SAIL to determine ing very specific questions the NMR solution structure about protein structure and of this 41-kDa maltodextrinbinding protein. dynamics."—STU B0RMAN C & E N / MARCH 6, 2006
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