Journal of Biomolecular NMR, 18: 23–31, 2000. KLUWER/ESCOM © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 23 Direct measurement of 1 H- 1 H dipolar couplings in proteins: A complement to traditional NOE measurements F. Tian, C.A. Fowler, E.R. Zartler, F.A. Jenney Jr., M.W. Adams & J.H. Prestegard ∗ Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602–4712, U.S.A. Received 7 April 2000; Accepted 22 June 2000 Key words: CT-COSY, 1 H- 1 H residual dipolar coupling, protein structure Abstract An intensity-based constant-time COSY (CT-COSY) method is described for measuring 1 H- 1 H residual dipolar couplings of proteins in weakly aligned media. For small proteins, the overall sensitivity of this experiment is comparable to the NOESY experiment. In cases where the 1 H- 1 H distances are defined by secondary structure, such as 1 H α - 1 H N and 1 H N - 1 H N sequential distances in α-helices and β-sheets, these measurements provide useful orientational constraints for protein structure determination. This experiment can also be used to provide distance information similar to that obtained from NOE connectivities once the angular dependence is removed. Because the measurements are direct and non-coherent processes, such as spin diffusion, do not enter, the measurements can be more reliable. The 1/r 3 distance dependence of directly observed dipolar couplings, as compared with the 1/r 6 distance dependence of NOEs, also can provide longer range distance information at favorable angles. A simple 3D, 15 N resolved version of the pulse sequence extends the method to provide the improved resolution required for application to larger biomolecules. Introduction Recent progress in the sequencing of genes is plac- ing increasing demands on the efficiency of structure determination methodology, whether by X-ray crystal- lography or NMR (Sali and Kuriyan, 1999). For NMR, some additional challenges exist in that some of the proteins coded by genes will not express or refold at levels suitable for the labeling required in most triple resonance experiments. These facts suggest reconsid- eration of the value of NMR experiments that require little ( 15 N only) or no isotopic labeling and incorporate new sources of structural information such as resid- ual dipolar couplings. We consider here an experiment that measures 1 H homonuclear dipolar couplings and compare its performance to a traditional NOE experi- ment on two small proteins at natural abundance and labeled only with 15 N. Data from both a 2D homonu- ∗ To whom correspondence should be addressed. E-mail: jpresteg@ccrc.uga.edu clear version and a 3D 15 N resolved version of the experiment are presented. Measurement of inter-proton NOEs is widely recognized as the foundation of biomolecular structure determination by NMR (Wüthrich, 1986), yet there are several reasons that we should consider replacing or supplementing these experiments. For example, NOEs in large molecules suffer from spin diffusion, mak- ing their quantitative interpretation difficult. The steep distance dependence of NOEs makes relationships be- tween remote parts of molecules hard to determine, and the basic dependence of NOEs on both correla- tion time and distance complicates interpretation in the presence of anisotropic or internal motions. A possible source of information to supplement NOEs is the direct measurement of dipolar couplings in oriented media (Tolman et al., 1995; Tjandra and Bax, 1997; Clore et al., 1998; Prestegard, 1998). Sev- eral potential advantages are associated with the use of dipolar coupling data. Because dipolar coupling mea- surements are direct, non-coherent transfer processes, such as spin diffusion, do not complicate interpreta-