An approach for high-throughput structure determination of proteins by NMR spectroscopy.
An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing 13C-methyl labeled Val, Leu, and Ile (delta1) and protonated Phe and Tyr in a deuterated background. Using this strategy, the key NOEs that define the hydrophobic core and overall fold of the protein are easily obtained. NMR data are acquired using cryogenic probe technology which markedly reduces the spectrometer time needed for data acquisition. The approach is demonstrated by determining the overall fold of the antiapoptotic protein, Bcl-xL, from data collected in only 4 days. Refinement of the Bcl-xL structure to a backbone rmsd of 0.95 A was accomplished with data collected in an additional 3 days. A distance analysis of 180 different proteins and structure calculations using simulated data suggests that our method will allow the global folds of a wide variety of proteins to be determined.
Medek
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Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, IL 60064, USA.
Olejniczak
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Meadows
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Fesik
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D000465:Algorithms; D002247:Carbon Isotopes; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry; D012680:Sensitivity and Specificity; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D002247:Carbon Isotopes; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein