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 2 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 NÀH···N [1][2][3] and NÀ H···O = C [4][5][6] 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 À H···N hydrogen bonding in nucleic acids [7,8] or NÀH···O=C bonds in proteins, [9] the intrinsic through-H-bond nature of the scalar coupling is appealing. J-based correlation spectroscopy [10,11] has been demonstrated for proteins in the solid state [12][13][14][15] and has been successful in measuring J couplings down to a few Hertz, including N À H···N H-bond couplings in crystals of organic molecules [16][17][18] . The small values for the scalar coupling constants of Hbonds in proteins, with average values in ubiquitin for NÀ H···O=C H-bond scalar coupling constants ( 3h J N,C' ) of (À0.38 AE 0.12) Hz (a-helix) to (À0.65 AE 0.14) Hz (b-sheet), [4] make these experiments challenging in terms of sensitivity. In liquids, difficulties arise particularly for larger molecules, where the coherence decay owing to faster T 2 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 T 2 ') include the use of high magic-angle-spinning (MAS) frequencies [19] and the dilution of the 1 H spin system by extensive deuteration [15,20] 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 3h J NC' couplings, and were recorded on a microcrystalline sample of 2 H, 13 C, 15 N-labeled ubiquitin, which was protonated at 20 % of the backbone...