In sequence-function investigations, approaches are needed for rapidly screening protein variants for possible changes in conformation. Recent NMR methods permit direct detection of hydrogen bonds through measurements of scalar couplings that traverse hydrogen bonds (trans-H-bond couplings). We have applied this approach to screen a series of five single-site mutants of the sweet protein brazzein with altered sweetness for possible changes in backbone hydrogen bonding with respect to wild-type. Long-range, three-dimensional HNCO data were collected from the six brazzein proteins labeled uniformly with carbon-13 and nitrogen-15. In wild-type brazzein, this approach identified 17 backbone hydrogen bonds. In the mutants, altered magnitudes of the couplings identified hydrogen bonds that were strengthened or weakened; missing couplings identified hydrogen bonds that were broken; and new couplings indicated the presence of new hydrogen bonds. Within the series of brazzein mutants investigated, a pattern was observed between sweetness and the integrity of particular hydrogen bonds. All three “sweet” variants exhibited the same pattern of hydrogen bonds, whereas all three “non-sweet” variants lacked one hydrogen bond at the middle of the -helix, where it is kinked, and one hydrogen bond in the middle of -strands II and III, where they are twisted. Two of the “non-sweet” variants lack the hydrogen bond connecting the N- and C-termini. These variants showed greater mobility in the N- and C-terminal regions than wild-type brazzein.