Protein side-chain rotamers from dipolar couplings in a liquid crystalline phase.

One-bond backbone dipolar couplings can readily be measured for proteins dissolved in a dilute liquid crystalline phase.1-3 They can be used to refine NMR structures determined by conventional methods or, in favorable cases, be sufficient to determine a structure de novo.4,5 They also provide an invaluable tool for quality evaluation.6,7 Nearly all focus thus far has been on the measurement of backbone dipolar couplings. However, side-chain conformations determine the critical details of the protein surface, and their characterization is therefore of the utmost importance. Here, we demonstrate a simple and sensitive method for measuring Câ-Hâ dipolar couplings. Because for staggered rotamers the Câ-Hâ bonds are parallel to either CR-HR, CR-N, or CR-C′ bonds, comparison of the dipolar couplings frequently can identify the side chain rotamer directly, even in the absence of a backbone structure. As shown below, the CR-HR coupling is obtained from the same experiment as the Câ-Hâ coupling. The CR-N dipolar coupling is not easily measured at high accuracy,8 but to a good approximation this bond is parallel to the CR-C′ bond of the preceding residue (rmsd ) 6.5°, as derived from high-resolution crystal structures). For residues with a Câ methylene site, our method measures the sum of the two Câ-Hâ couplings. For a staggered rotamer this sum must be equal to the normalized sum of CR-HR and CR-N (ø1 ) -60°), CR-HR and CR-C′ (ø1 ) 180°) or CR-N and CR-C′ couplings (ø1 ) +60°). The CR-HR and Câ-Hâ couplings are measured with the CB(CA)CONH pulse scheme9 of Figure 1. It has been slightly modified from its original implementation by the use of adiabatic pulses, which decrease effects of RF inhomogeneity and offset, thereby significantly increasing sensitivity.10 The experiment is performed three times, in an interleaved manner, with the 1H 180° pulse during t1 evolution switched between positions a, b, and c. The JCH rephasing of 13Câ (and 13CR) at the end of the constanttime t1 evolution period depends on the duration of ∆1. Ignoring cross-correlated relaxation during the short constant-time evolution period, the ∆1-dependence of the methine and methylene CR and Câ signal intensities is given by