An approach for high-throughput structure determination of proteins by NMRspectroscopy

An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing C-13-methyl labeled Val, Leu, and Ile (de lta1) and protonated Phe and Tyr in a deuterated background. Using this str ategy, the key NOEs that define the hydrophobic core and overall fold of th e protein are easily obtained. NMR data are acquired using cryogenic probe technology which markedly reduces the spectrometer time needed for data acq uisition. The approach is demonstrated by determining the overall fold of t he antiapoptotic protein, Bcl-xL, from data collected in only 4 days. Refin ement of the Bcl-xL structure to a backbone rmsd of 0.95 Angstrom was accom plished with data collected in an additional 3 days. A distance analysis of 180 different proteins and structure calculations using simulated data sug gests that our method will allow the global folds of a wide variety of prot eins to be determined.