Site-Specific Hydrogen Exchange in a Membrane Environment Analyzed by Infrared Spectroscopy

Hydrogen exchange is a powerful method to examine macro-molecules. In membrane proteins, exchange can distinguish between solvent accessible and inaccessible residues due to shielding by the hydrophobic environment of the lipid bilayer. Herein, rather than examining which residues undergo hydrogen exchange, we employ a protocol that enables the full deuteration of all polar hydrogens in a membrane protein. We then measure the impact of hydrogen exchange on the shift of the amide I vibrational mode of individually labeled sites. The results enable us to correlate polarity with vibrational shifts, thereby providing a powerful tool to examine specific locations within a membrane protein in its native membrane environment.