Direct detection of (3h)J(NC') hydrogen-bond scalar couplings in proteins by solid-state NMR spectroscopy.

Hydrogen bonds (H-bonds) are important and ubiquitous interactions in chemistry and biology. They are a key element in proteins and nucleic acids for stabilizing the three-dimensional fold and are thus important for the functionality. The presence of an H-bond can be indirectly deduced from the local geometry as obtained from X-ray or NMR methods, and a variety of NMR parameters depend also on hydrogen bonding (e.g. chemical shifts induced by H-bonds or H quadrupolar coupling constants). Direct evidence of hydrogen bonding, however, is provided by the presence of an H-bond-mediated scalar coupling. Experiments that directly measure J couplings across N H···N and N H···O=C bonds in nucleic acids and proteins, respectively, have been introduced for solution-state NMR spectroscopy and have received great interest. Such experiments allow the direct identification of the donor and acceptor side of each Hbond, as well as the determination of the size of the coupling—which is a very sensitive probe of the geometry around an H-bond. Even though solid-state NMR experiments can also use the dipolar interaction to indirectly probe N H···N hydrogen bonding in nucleic acids 8] or N H···O=C bonds in proteins, the intrinsic through-H-bond nature of the scalar coupling is appealing. J-based correlation spectroscopy has been demonstrated for proteins in the solid state and has been successful in measuring J couplings down to a few Hertz, including N H···N H-bond couplings in crystals of organic molecules . The small values for the scalar coupling constants of Hbonds in proteins, with average values in ubiquitin for N H···O=C H-bond scalar coupling constants (JN,C’) of ( 0.38 0.12) Hz (a-helix) to ( 0.65 0.14) Hz (b-sheet), make these experiments challenging in terms of sensitivity. In liquids, difficulties arise particularly for larger molecules, where the coherence decay owing to faster T2 relaxation greatly attenuates the signal during the long time periods required for the polarization transfer mediated by the small J coupling. The situation is even more challenging in solids because of the presence of secular anisotropic interactions, which provide additional mechanisms of transverse dephasing. The strategies employed to minimize this additional dephasing (and thus minimizes T2’) include the use of high magic-angle-spinning (MAS) frequencies and the dilution of the H spin system by extensive deuteration 20] to avoid the need for high-power proton decoupling over extended time periods. Herein, we demonstrate the use of a deuterated and partially backprotonated microcrystalline protein at high MAS frequencies, to directly measure sub-Hertz trans-Hbond scalar coupling constants. Figure 1 shows strips from a “long-range” 3D HNCO correlation experiment optimized for the detection of JNC’ couplings, and were recorded on a microcrystalline sample of H,C,N-labeled ubiquitin, which was protonated at 20 % of the backbone amides and other exchangeable sites. The pulse sequence used to obtain these data is a purely Jbased HNCO experiment, akin to an experiment proposed by Cordier and Grzesiek for measurement of JNC’ in the solution state. The details of the pulse sequence are shown in Figure S1 of the Supporting Information. The key features of this H-detected experiment are long out-and-back N C’ INEPT blocks for a duration of 2T= 66.6 ms to transfer coherence from the H-bond donor N to the acceptor C’, and was carried out in the presence of low-power (3.1 kHz) H decoupling. The evolution resulting from the large JNC’ coupling (approximately 15 Hz = 1/(2T)) vanishes at multiples of 2T. Therefore, the small JNC’ couplings become detectable and lead to coherence transfer over H-bonds. The data in Figure 1 unambiguously show cross-peaks that arise from transfer over N H···O=C hydrogen bonds involving the amides of residues L15, V17, I44, K6, and L50 (the latter two have overlapping cross-peaks) with the carbonyl groups of I3, M1, H68, L67, and L43. The size of the couplings involved is known to be 0.5–0.6 Hz (see below), which is almost an order of magnitude smaller than the smallest couplings measured to date using solid-state NMR spectroscopy. To confirm the results and to investigate the reproducibility of the measurement, we have performed an additional experiment under slightly different conditions and using a sample with a higher degree of protonation on the exchangeable sites (30 % rather than 20%). The 2D long-range H(N)CO data set obtained on this sample is shown in Figure S2 of the Supporting Information. The spectrum reveals cross-peaks arising from H-bonds of residues F4 and E34, in addition to the ones observed in the 3D spectrum. In the 3D data set of Figure 1 these cross-peaks overlapped with residual one-bond cross-peaks and are resolved in the [*] Dr. P. Schanda, M. Huber, Dr. R. Verel, Dr. M. Ernst, Prof. Dr. B. H. Meier Physical Chemistry, ETH Z rich Wolfgang-Pauli-Strasse 10, 8093 Z rich (Switzerland) Fax: (+ 41)44-632-1621 E-mail: pasa@nmr.phys.chem.ethz.ch beme@ethz.ch Homepage: http://www.ssnmr. ethz.ch