Local knowledge helps determine protein structures.

T he nuclear Overhauser effect (NOE) has been the workhorse of structural studies of macromolecules in solution by NMR spectroscopy. The NOE is a measure of the rate of magnetization transfer between nuclei, induced by modulation of the magnetic dipole–dipole coupling between nuclear spins by overall tumbling of the molecule in solution (1). The efficiency of magnetization transfer via this mechanism is low; consequently, NOEs are typically only observed for protons within 5 Å, and lengthy experiments are required in order to detect the weak effect. NOE magnitudes provide a ‘‘spectroscopic ruler’’ through the approximate r 6 dependence on the distance between proton pairs. Although it takes surprisingly few such distance estimates to determine the overall fold of a protein (2)—provided the estimates are distributed evenly throughout the protein—even a complete set of NOEs yields far fewer constraints on the structure than are required to determine all 3N 6 degrees of freedom for an Natom protein. The fold is even more highly underdetermined when protein flexibility is taken into account. By complementing NMR measurements with prior knowledge of protein structure, usually in the form of a potential energy function that describes the physical plausibility of a model structure, NMR has emerged as a valuable complement to x-ray crystallography for structure determination. The Protein Data Bank (PDB) (3) now contains 7,000 NMR protein structures, and 1,000 new structures are anticipated to be added in 2008. One way to further hasten the rate of protein structure determination by NMR is to develop alternatives that avoid the need to measure and assign NOEs. In this issue of PNAS, Shen et al. (4) describe a viable alternative to NOE-based structure determination for small proteins. Nevertheless, reliance on NOEs for determination of biomolecular structure presents real challenges. A NOE must be assigned to two specific nuclei before it can be used to constrain a structural model, and assignment becomes more challenging as the size of the molecule increases. Even a modest-sized protein can yield many thousands of detectable NOEs. NOEs are frequently missing from flexible regions, and NOEs that can be measured are usually highly correlated with one another. The information content of all NOEs is not the same: NOEs between residues that are distant in the primary sequence (called ‘‘long-range NOEs’’) are more important for constraining the overall fold than are NOEs between neighboring residues (‘‘short-range NOEs’’) or intraresidue NOEs that provide no additional structural information (‘‘redundant NOEs’’). The latter two types of NOEs are far more prevalent. The confidence level in an assignment is greater when a NOE is correlated with other assigned NOEs, but distinguishing a unique longrange NOE from an erroneous assignment can be challenging. Indeed, the small number of demonstrably incorrect NMR structures reported can nearly all be attributed to a handful of erroneous NOE assignments. NOEs are by no means the only NMR-observable parameters that are sensitive to structure. Vicinal couplings, residual dipolar couplings, and chemical shifts are all sensitive to structure and are all routinely used to supplement NOE-derived distance restraints in protein structure determination (5). Chemical shifts are the easiest to measure and are the first parameters to be determined in assigning resonances to specific nuclei. Chemical shift is a measure of the Larmour precession frequency of a magnetic spin under the influence of the local magnetic field (6). The field experienced by a nucleus is perturbed from the applied field strength of the instrument by the distribution of electrons near the nucleus, which can either diminish or augment the applied field. As the name implies, chemical shift is sensitive to the number and nature of substituents covalently connected to the nucleus, and to their conformation. The correlation between 1H, 13C, and 15N chemical shifts of the polypeptide backbone and backbone dihedral angles has long been viewed as a potential alternative to NOEs for determining solution structure, but the imprecision of the correlation has proved limiting. Nevertheless, parallel progress in improving the correlation between chemical shifts and structure (both quantum mechanical and empirical) and de novo methods for protein structure prediction led Wishart and Case (7) to anticipate in 2001 that a combination of chemical shifts and structure prediction methods would be-