Pressure alters electronic orbital overlap in hydrogen bonds

Pressure-induced changes in (3h)J(NC)' scalar couplings through hydrogen bonds were investigated in the immunoglobulin binding domain of streptococcal protein G. H-1, N-15 and C-13 triple-resonance NMR spectroscopy coupled with the on-line high pressure cell technique was used to monitor (3h)J(NC)' scalar couplings at 30 and 2000 bar in uniformly labeled N-15 and C-13 protein isotopes. Both increased and decreased (3h)J(NC)' scalar couplings were observed at high pressure. No correlation with secondary structure was apparent. The difference in coupling constants as well as pressure-induced chemical shift data suggests a compaction of the helix ends and an increase of the helix pitch at its center in response to pressure. Our data provides the first direct evidence that the electronic orbital overlap in protein backbone hydrogen bonds is altered by pressure.