NMR detection of side chain-side chain hydrogen bonding interactions in C-13/N-15-labeled proteins

We describe the direct observation of side chain-side chain hydrogen bondin g interactions in proteins with sensitivity-enhanced NMR spectroscopy. Spec ifically, the remote correlation between the guanidinium nitrogen N-15(epsi lon) of arginine 71, which serves as the hydrogen donor, and the acceptor c arboxylate carbon (CO2gamma)-C-13 of aspartate 100 in a 12 kDa protein, hum an FKBP12, is detected via the trans-hydrogen bond (3h)J(N epsilon CO2 gamm a) coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The ( 3h)J(N epsilon CO2 gamma) coupling constant appears to be even smaller than the average value of backbone (3h)J(NC') couplings, consistent with more e xtensive local dynamics in protein side chains. The identification of trans -hydrogen bond J-couplings between protein side chains should provide usefu l markers for monitoring hydrogen bonding interactions that contribute to t he stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.